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Doctoral Thesis

Metabolic studies of microorganisms using fractional 13C-labeling and 2D NMR

Author(s): Hochuli, Michel

Publication Date: 1999

Permanent Link: https://doi.org/10.3929/ethz-a-003822562

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ETH Library

Diss. ETHNr. 13307

Metabolic studies of microorganisms using

fractional 13C-IabeIing and 2D NMR

A dissertation submitted to the

Swiss Federal Institute of Technology Zürich

for the degree of

Doctor of Natural Sciences

presented by

Michel Hochuli

Dipl. Chem. Universität Bern

born on October 19, 1971

citizen of Re l tri au (Aargau)

accepted on the recommendation of

Prof. Dr. Kurt Wiithnch, Referent

Prof. Dr. Thomas Leismger, Korreferent

Prof. Dr. Thomas Szypcrski. Korreferent

1999

r. >>,

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n il

3

Vorwort

Mein Dank geht zuerst an Herrn Prof. Kurt Wüthrich. Er ermöglichte mir, diese Arbeit in

seiner Forschungsgruppe am Institut für Molekularbiologie und Biophysik der ETH Zürich

unter besten Voraussetzungen durchzuführen, und gewährte mir immerzu weitreichende

Unterstützung. Herrn Prof. Thomas Leisinger möchte ich für das durch die spontane

Uebernahme des Korreferats bezeugte Interesse an dieser Arbeit herzlich danken.

Für die sehr fruchtbare Zusammenarbeit schulde ich Herrn Prof. Thomas Szyperski ganz

besonderen Dank. Er führte mich m die hier benutzte Technik zur Stoffwechseluntersuchung

ein, und hat mir während der ganzen Dissertaüonszeit als Betreuer stets grosse Hilfe und

Unterstützung entgegengebracht.

Besonderer Dank gebührt auch Herrn Dr. Ralf Glaser. Er schrieb ein Computerprogramm,

welches die Auswertung der NMR-Spcktren für die hier präsentierten

Stoffwechseluntersuchungen wesentlich erleichterte. Für die Zusammenarbeit bei der

Untersuchung der Aminosäuresynthesewege im halophilen Arch aeon Haloarcula hispanica

bin ich Herrn Prof. Dieter Oestcrhelt (MPI für Biochemie, München) und Herrn Prof. Heiko

Patzelt (Sultan Qaboos University, Oman) zu grossem Dank verpflichtet. Weiter möchte ich

Herrn Dr. Uwe Sauer, Jocelyne Fiaux, Marcel Emmerling und Michael Dauner für die

wertvolle Zusammenarbeit und die vielen interessanten Diskussionen in Stoffwechselfragen

herzlich danken.

Für eine Vielzahl interessanter Gespräche und ihre Hilfe im Forschungsalltag danke ich

allen Kollegen im Institut für Molekularbiologie und Biophysik, im besonderen Herrn Daniel

Braun, PD Rene Brunisholz, Dr. Fred Damberger. Dr. César Fernandez, PD Peter Güntert, PD

Gerhard Wider, Dr. Roland Rick, Dr. Michael Salzmann und Dr. Reinhard Wimmer.

Herzlichen Dank auch an Rudolf Baumann. Er war für das Lösen von computer¬

technischen Problemen stets mit Rat und Tat zur Stelle.

Herrn Prof. Kurt Wüthrich möchte ich noch einmal herzlich dafür danken, dass er mir die

Möglichkeit bot, parallel zu meinem Doktorat die erste Vorprüfung für Aerzte an der

medizinischen Fakultät der Universität Zürich abzulegen.

Reto Bader und Jocelyne Fiaux danke ich für das kritische Durchlesen des Manuskripts

meiner Dissertation.

Meinen Eltern Erwin und Regula danke ich ganz herzlich dafür, dass sie meine

Ausbildung ermöglicht haben.

c>:-:-ftT • uer /\

leaf

5

Summary

Biosynthetically-directed fractional C-labeling of proteinogenic amino acids

combined with two-dimensional [nC,'ill-correlation nuclear magnetic resonance

(NMR) spectroscopy (2D [ C, H]-COSY) for the analysis of the non-random

13C-labehng patterns in the amino acids is an efficient method for metabolic studies of

microorganisms. In this approach, contiguous carbon fragments arising from a single

carbon source molecule are traced through a cellular bioreaction network. Since the

patterns of intact carbon fragments observed for a given metabolite very often depend

on which pathway was used for its synthesis, information about both active

biochemical pathways and metabolic flux ratios is obtained. This methodology was

applied to investigate amino acid biosynthesis pathways in the extremely halophilic

archaeon Haloarcida hispanica, and to study the central carbon metabolism of

Escherichia coli BL21(DE3) growing in D20-containing minimal media.

Biosynthesis of proteinogenic ammo acids in Haloarcida hispanica was explored

using biosynthetically-directed fractional C-labeling with a mixture of 90%

unlabeled and 10% uniformly C-labeled glycerol as the only carbon source in a

minimal medium. The experimental data provided evidence for a split pathway for

isoleucine biosynthesis, with 56% of the total isoleucine originating from threonine

and pyruvate via the threonine pathway, and 44% from pyruvate and acetyl-CoA via

the pyruvate pathway. In addition, the diaminopimelate pathway involving

diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis, and the

analysis of the 13C-labeling pattern in the aromatic ring of tyrosine indicated novel

biosynthetic pathways, which have not been further characterized within the scope of

the thesis. For the other evaluated proteinogenic amino acids, the data were consistent

with commonly found biosynthetic pathways. The comparison with the amino acid

metabolisms of eucarya and bacteria supports the theory that most of the pathways for

the synthesis of proteinogenic amino acids were probably established before ancient

cells diverged into archaea, bacteria and eukarya.

6

Isotope effects on the flux distribution in central carbon metabolism due to the

addition of different amounts of D20 (0 to 70 %) to the M9 minimal medium were

investigated for Escherichia coli BL21(DE3) cells during exponential growth, using

biosynthetically-directed fractional labeling with a mixture of 70 % unlabeled and

30 % uniformly C-labeled glucose as the sole carbon source. With the

aforementioned growth conditions the E. coli cells show only very limited response to

the stress imposed by the presence of D20 in the growth medium. The topology of the

active biosynthetic pathways remained unchanged at elevated D20 content. With

regard to the three main processes, the metabolic flux ratios characterizing glycolysis

and the pentose phosphate pathway were not measurably affected by the addition of

D20, but at higher D20 contents the anaplerotic supply of the tricarboxylic acid cycle

via carboxylation of phosphoenolpyruvate increased relative to the influx of acetyl-

CoA. Furthermore, the addition of D20 affected the C\ metabolic pathways generating

serine and glycine. Cells that had been adapted for growth in D20 exhibited the same

response to the presence of D20 in the nutrient medium as non-adapted cells.

Implications of these data on the preparation of recombinant deuterated proteins for

NMR studies are discussed.

7

Zusammenfassung

Biosynthetisch gesteuerte partielle C-Markierung der Aminosäuren, kombiniert

1 ° 1 1 'X Îmit zweidimensionaler [ C, H]-NMR-Korrelationsspektroskopie ([ C, H]-COSY)

für die Analyse der resultierenden, nicht-zufällig verteilten C-Markierungen, ist eine

rationelle Methode für Stoffwechseluntersuchungen in Mikroorganismen. Dabei

verfolgt man intakte C2 oder C^ Fragmente aus dem Gerüst der Kohlenstoffquelle im

Stoffwechselnctzwerk der Zelle. Da die für einen bestimmten Metaboliten

beobachteten Muster der intakten Fragmente davon abhängen, über welchen Weg

dieser synthetisiert worden ist, lässt sich mit dieser Methode untersuchen, welche

Stoffwechselwege unter vorgegebenen Bedingungen aktiv sind, und es können

Flussverhältnisse zwischen verschiedenen Reaktionswegen im Stoffwechselnetzwerk

berechnet werden. In der vorliegenden Arbeit wurde diese Methode angewandt, um die

Aminosäurebiosynthesewege im extrem halophilen Archaeon Haloarcida hispanica

zu erforschen, und um den Zentralstoffwechsel von Escherichia coli BL21(DE3)

Zellen während des Wachstums in D20-enthaltenden Medien zu studieren.

Für die Untersuchung der Aminosäurebiosynthesewege wurde Haloarcida

hispanica in einem Minimalmedium mit 90% unmarkiertem and 10% vollständig

C-markiertem Glycerol als einziger Kohlenstoffquel le aufgezogen. Die erhaltenen

Daten zeigten, dass Isoleucin parallel über zwei verschiedene Wege gebildet wird,

einerseits zu 56 % aus den Vorlaufermolekülen Threonin und Acetyl-CoA über den

Threoninweg, und andererseits zu 44 % aus Pyruvat und Acetyl-CoA über den

Pyruvatweg. Zusätzlich stellte sich heraus, class Lysin über das Enzym

Diaminopimelatdehydrogenase im Diaminopimelatwcg synthetisiert wird, und die

l3C-Markierungsmustcr im aromatischen Ring von Tyrosin wiesen daraufhin, dass die

Tyrosinsynthese in H. hispanica über einen noch unbekannten Weg verläuft, der in der

vorliegenden Arbeit aber nicht weiter charakterisiert wurde. Für die anderen

untersuchten Aminosäuren stimmten die Daten mit den sonst üblicherweise

gefundenen Synthesewegen überein. Ein Vergleich des Aminosäurestoffwechsels der

Archaea mit demjenigen von Bacteria und Eukarya lässt den Schluss zu, dass die

8

meisten Aminosäuresynthesewege wahrscheinlich schon vor der Aufspaltung der

Organismen in die Domänen der Archaea, Bacteria und Eukarya existierten.

Um Deuterium-Isotopcneffekte auf die Flussverteilung im Zentralstoffwechsel von

E. coli BL21(DE3) Zellen zu untersuchen, die exponentiell in D20-enthaltenden

Medien wachsen, wurden die Zellen in M9 Minimalmedien mit verschiedenem

D20-Gehalt (0 bis 70 %) gezüchtet, wobei eine Mischung aus 70 % unmarkierter und

î °

30 % vollständig C-markierter, protonierter Glukose als einzige Kohlenstoffquelle

eingesetzt wurde. Die Daten zeigten, dass E. coli Zellen unter den gegebenen

Wachstumsbedingungen auf den durch die verschiedenen kinetischen und

thermodynamischen Isotopeneffekte ausgelösten Stress nur sehr beschränkt reagieren.

In D20-haltigen Medien wurden keine zusätzlichen Reaktionswege aktiviert oder

abgeschaltet, und die Flussverhältnisse in der Glykolyse und im Pentose-Phosphatweg

änderten sich nicht signifikant wenn D20 zum Medium gegeben wurde. Im Gegensatz

dazu nahm bei höherem D20-Gchalt im Medium die anaplerotische Versorgung des

Zitronensäurezyklus über die Karboxylicrung von Phosphoenolpyruvat zu relativ zum

Zustrom von Acetyl-CoA. Zudem beeinflusste D20 den C [-Stoffwechsel, aus dem

Serin und Glycin synthetisiert werden. Zellen, die für das Wachstum in D20-

enthaltenden Medien adaptiert worden waren, zeigten im Vergleich zu nicht

adaptierten Zellen keinen Unterschied in den Flussverhältnissen. Die Auswirkungen

dieser Daten auf die Herstellung von rekombinanten, deutenerten Proteinen für NMR-

Untersuchungen werden diskutiert.

9

Table of Contents

Vorwort 3

Summary 5

Zusammenfassung 7

Abbreviations 11

1. Introduction 13

1.1. The fractional carbon-13 labeling approach for metabolic studies 17

2. Amino acid biosynthesis in the halophilic archaeon

Haloarcula hispanica 25

2.1. Isoleucine biosynthesis 32

2.2. Lysine biosynthesis 39

2.3. Tyrosine biosynthesis 44

2.4. Enzymatic cleavage of surplus threonine 48

2.5. Discussion 50

3. Effects of deuterium oxide on the central carbon metabolism

of Escherichia coli 56

4. Literature 78

5. Appendix 89

5.1. Amino acid biosynthesis in Haloarcida hispanica 89

10

5.2. Effects of deuterium oxide on the central carbon metabolism of

Escherichia coli 93

Cuniculum vitae 101

Publications 103

Abbreviations

ID, 2D 1-dimensional, 2-dimensional

COSY correlation spectroscopy

HSQC Heteronuclear single-quantum correlation

INEPT Insensitive nuclei enhanced by polarization transfer

NMR Nuclear magnetic resonance

ppm parts per million

TCA Tricarboxylic acid cycle

TOCSY Total correlation spectroscopy

TPPI Time proportional phase incrementation

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f S»ïà ^.mi |>

13

1. Introduction

Cellular metabolism is a highly integrated network of enzyme catalysed reactions,

by which the cell synthesizes its fundamental chemical ingredients, and by which it

obtains its energy (Stryer, 1996; Voet and Voet, 1995). In Escherichia coli for instance,

the network is composed of more than a thousand chemical reactions (Neidhardt et al,.

1996). These are organized into series of consecutive enzymatic reactions, called the

metabolic pathways. The reactants, intermediates and products of metabolic pathways

are referred to as metabolites. When the cell grows, nutrients from the environment are

taken up into the cell and transformed into a wide variety of cell constituents. In the

catabolic pathways, the nutrient molecules are exergonically broken down to simpler

intermediates, which are then further metabolized to a few end products. The catabolic

pathways provide the chemical energy and the building blocks for the anabolic

pathways, through which occurs the biosynthesis of the different molecular

components of cells such as nucleic acids, proteins, polysaccharides and lipids. In a

switchboard-like fashion, the cell directs the distribution and processing of metabolites

throughout this extensive map of pathways. The rates of reactions are tightly regulated

and coordinated by elaborate control mechanisms, so as to meet energy requirements

and to satisfy demands for biomass (Alberts et al. 1994; Martin, 1997; Newsholme and

Start, 1973). The cell can adapt the regulation to respond to varying environmental

conditions. Mutations of many kinds can even eliminate particular reaction pathways

and yet the cell survives, provided that certain minimum requirements are met.

A knowledge of cell metabolism is essential for understanding the biochemistry

and physiology of microbial growth, both out of an interest in the living system itself,

and for possible applications of microbiology in biotechnology and medicine (Bailey

and Ollis, 1986; Madigan et al., 1997; Neidhardt et al, 1990). Many important

practical consequences of microbial growth, such as infectious diseases, the production

of (specialty) chemicals, food additives and pharmaceutical products by

microorganisms, or the biodégradation of xenobiotics are linked to microbial

metabolism. Knowledge of metabolism aids in developing laboratory procedures for

14 Introduction

cultivating microorganisms, and in developing suitable procedures for preventing the

growth of unwanted microbes. Furthermore, this understanding is at the basis of all

attempts to rationally redesign cellular metabolism in order to optimize living systems

for biotechnological applications, a procedure which has been coined 'metabolic

engineering' (Bailey, 1991; Lessard, 1996: Stephanopoulos, 1998). Metabolic studies

cover different aspects such as (i) the elucidation of the sequences of reactions and the

topological structure of the network, i.e., the locations of nodes at which one substance

is either a substrate of two branching reactions or a product of two converging

reactions, (ii) the mechanisms of enzymatic reactions (Fersht, 1985; Kuby, 1991), (iii)

the (in vivo) thermodynamics (Jones, 1979) and kinetics of the reactions (Liao and

Lightfoot, 1988), and (iv) the control mechanisms that regulate the flow of metabolites

through the pathways.

Many different, well-established experimental techniques are available to explore

cellular metabolism. One can use inhibitors to block metabolic pathways at specific

enzymatic steps and study the effect on growth and production of metabolic

intermediates. Insight can be gained from the work with auxotrophic mutants, which

have one or several enzymes in a pathway inactivated or deleted and require the

pathway's product for growth, or one can identify purified metabolites and characterize

the enzymes that catalyse their interconversions. In particular, isotope tracer

experiments to follow the path of atoms and molecules through the metabolic network

have become routine. Among those, carbon isotope labeling protocols have

predominantly been employed because they provide direct insight into the carbon

metabolism of the cell.

Over time, a wealth of information has accumulated about the biochemistry,

regulation and genetics of individual enzymes or pathways in certain model microbes

such as Escherichia coli (Neidhardt et al., 1996), Bacillus subtilis (Sonenshein et al.,

1993) and Saccharomyces cerevisiae (Rose and Harrison, 1989; Strathern et al., 1982).

However, although this large body of information on the single network components is

available, the regulatory response of the integral metabolic network, i.e. changes in

carbon flux through the different reactions and activation of pathways in response to

varying environmental conditions or to genomic manipulations, remains difficult to

predict. Therefore, experimental methods to measure or estimate the in vivo flux

15

distribution, and to assess the actual topology of the metabolic network are of great

value to understand the interplay of reactions, and complement bioinformatics and

mathematical modeling (Bailey, 1998; Fell, 1997; Hatzimanikatis et al., 1995;

Schlosser et al., 1994) for a more systematic approach in the improvement of metabolic

functions for practical applications (Hggeling et al., 1996; Stephanopoulos, 1999;

Stephanopoulos and Sinskey, 1993; Varma and Palsson, 1994; van Gulik and Heijnen,

1995).

The value of 13C4abeling experiments for providing information on active

biochemical pathways and on intracellular flux distributions has long been recognized.

(Cerdan and Seclig, 1990; Gadian, 1995: London, 1988; Szyperski, 1998; Walsh and

Koshland, 1984). The redistribution of the label in different metabolites or biosynthesisI ~*

products can be assessed by C-NMR spectroscopy or in mass spectra (Rosenblatt et

al., 1992; Christensen and Nielsen, 1999), the primary observables being the positional

13C enrichments (Cerdan and Seelig, 1990; Scott and Baxter, 1981) or the isotopomer

distributions (Jeffrey et al., 1991; London, 1975). Selectively C-labeled substrates

for 13C-labeling of amino acids, monitored either as intracellular metabolites, as

extracellular products, or in the cell protein, have been applied in several instances

(Ekiel et al., 1984; Ishino et al., 1991; Rollin et al., 1995), and most recently also in

combination with flux balancing (Marx et al.. 1996). However, methods based on

selectively 3C-labeled substrates have major drawbacks, since they usually rely on

numerous purification steps, and are expensive because large amounts of expensive

isotopically labeled substrates are required. 'Biosynthetically-directed fractional 13C~

labeling of proteinogenic amino acids' (Senn et al., 1989; Szyperski, 1995; 1998;

Szyperski et al., 1992; 1996, Wüthrich et al., 1992) constitutes an efficient analytical

tool to explore microbial metabolism. In this approach, a mixture of uniformly Re¬

labeled and unlabeled carbon source molecules is fed into a bioreaction network and

the resulting 13C-labeling patterns in the amino acids are analysed in 2D [^C^H]-

correlation NMR spectra of hydrolysed biomass, without prior separation of the amino

acids. This labeling strategy enables one to trace contiguous carbon fragments arising

from a single carbon source molecule through a cellular bioreaction network, which

yields a detailed picture of the breakdown of the precursor molecules in the bioreaction

network under consideration. Because the patterns of intact carbon fragments observed

16 Introduction

for a given metabolite very often depend on which pathway was employed for its

synthesis, one obtains information about both the actual topology of the bioreaction

network and flux ratios at several key points in central metabolism in a single

experiment (Fiaux et al, 1999; Sauer et al., 1997; 1999, Szyperski, 1995; 1998;

Szyperski et al, 1992; 1996). The experiment can be performed at moderate costs since

only a fraction of the employed carbon source needs to be C-labelcd.

1.1. The fractional carbon-13 labeling approach for metabolic studies 17

1.1. The fractional carbon-13 labeling approach for

metabolic studies

To achieve biosynthetically-directed fractional C-labeling, the cells are grown in

a minimal medium containing a single uniformly C-labeled carbon source diluted

with its non-enriched form, e.g. 10 % [ C6]-glucose and 90 % unlabeled glucose. The

isotope enrichment at all carbon positions is then uniform, and the ~C- C scalar

coupling fine structure remains the sole observable providing biosynthetic information.

The use of a uniformly C-labeled metabolic precursor enables one to trace C- C

connectivities in the bioreaction network by NMR and therefore corresponds to a

carbon-carbon bond labeling approach. Because intact two- or three carbon fragments

from the uniformly C-labeled carbon source are incorporated into the metabolites,

non-random C-labeling patterns are eventually observed in the amino acids. For

example, in a sample labeled with 10 % [ C6]-glucose and 90 % unlabeled glucose,

the probability to find two adjacent carbon atoms that are simultaneously C-labeled

is only 1.2 % if the two spins derive from different source molecules of glucose

(random C-labeling), whereas this probability rises to 10 % if the two spins are part

of a carbon fragment that derives from the same glucose molecule (non-random C-

labeling) (Fig. l.l). Hence, the O- C scalar coupling fine structure of the terminal

carbon atom in a C2-fragment, e.g., is dominated by a singulet for the case with random

C-labcling, because here the neighbouring carbon is mostly C, whereas a doublet

1 }dominates for non-random ~

C-labeling, because in this case the neighbouring carbon

is mostly C (Fig. 1.1).

Routinely, the C- C scalar coupling fine structures of the different amino acids

are assessed in a 2D [ C, H]-HSQC spectrum of completely hydrolyscdbiomass. This

experiment offers excellent sensitivity (Bodenhausen and Ruben, 1980), and a

separation of the amino acids prior to NMR analysis is not required, since all relevant

peaks are well resolved in the 2D NMR spectrum (Fig. 1.2A). The 13C-13C scalar

coupling fine structures are observed along the 13C axis (Fig. 1.2B).

18 Introduction

random l3C-labeling

the two spins derive from Relative

different source molecules of glucose abundance

13C-13C scalar couplingfine structure

(the encircled nucleus is observed)

9.9 ct

1.2 9o

H"

<o(t3C) [IIzl

1 ^non-random C-labeling

the two spins derive from

the same source molecule of glucose

i.l o/.

10%

/h;i

w(13C) [11/]

Figure 1.1: Biosynthetic fractional 13C-labeIing of amino acids with 10 %

[Q;]-glucose and 90 % unlabeled glucose.

The incorporation of intact C-C fragments from a single source molecule of glucose into the

amino acids yields non-random 'V-labeling patterns (see the text). The probability to find two

neighbouring carbon atoms is iO 9c when both carbon atoms derive from the same source

molecule, whereas it is only 1 2 7 if they derive from different source molecules. The intensity of

the individual .multiplet components in the nC tine structures reflect the relative abundances of

the corresponding isotopomeis. In this example, the terminal caibon atom is observed.

1.1. The fractional carbon-13 labeling approachfor metabolic studies 19

49

A3,CO!("C)

[ppm]

54

59 .

a-Asp

a-Ala

a-Leu

a-His

a-Phe a-Tyr

a-Met

a-Ser

a-Glu

a-Lys & a-Arg

(/-lie

a-Pro

u-Val

cx-Thr

R-Ser

4.4

to2(lH) [ppm]

i

4.0

B1 I

a -Val

I i i

a-Ile illi I S 1

a -Ala IUI

a -Asp

11 s

a -Glu

11 Hi 11

a-Ser<

Uli40 0 -40

(Bt(l3C) (HZ)

Figure 1.2: 2D [13C,lHl-HSQC spectrum of a fractional 13C-labeled amino acid

hydrolysate.

(A) Region containing the 13cu-'Ha cross peaks of all amino acids except glycine in a

hydrolysate of fractionally' ^C-labeled cellular protein (Szyperski et al., 1996).

(B) Cross sections taken along 0)\ ( C). showing the C- 'C scalar coupling fine structure of

selected peaks in (A).

The C- C scalar coupling fine structures represent a superposition of the

individual multiplet patterns of the different L'C isotopomers. weighted by their

relative abundance (Fig. 1.3). For most carbons, only the one bond coupling constants

3/-1 13/Jqç are sufficiently large to be resolved in the C dimension, and therefore the C

fine structure is only determined by the 13C-labeling pattern of the directly attached

carbons (Krivdin and Kalabin, 1989). Furthermore, the proteinogenic amino acids

exhibit sufficient chemical shift dispersion (Wüthrich, 1976) so that strong l3C-13C

scalar coupling effects do not have to be considered for data interpretation when using

a modern high-field NMR spectrometer (Szyperski. 1995). For a central carbon in a

C3-fragment that exhibits different 1JC-K1C scalar coupling constants with the two

attached carbons, the l3C fine structure is composed of four different multiplet

20 Introduction

components (Fig. 1.3). For the C carbon of aspartate, e.g., it is composed of (i) a

singlet from the isotopomer where only the observed carbon is 13C-labeled, (ii) a

doublet with a small coupling constant (~ 35 Hz) from the isotopomer with C at Ca

and C", (iii) a doublet with a large coupling constant (~ 55 Hz) from the isotopomer

with 13C at C(X and C, and (iv) a doublet of doublets from the isotopomer where all

carbons in the C'-Ca~C^ fragment are C-labeled. According to the principle shown

in Fig. 1.1, the relative abundance of the individual C isotopomers and the intensity

of the corresponding multiplet components depend on the incorporation of intact

carbon fragments from the carbon source into the metabolites. Hence, the relative

multiplet intensities observed in the 13C fine structure can be translated to the relative

abundances of intact carbon fragments if values, see also Box 2.1), using probabilistic

equations (Szyperski, 1995). This yields the fractions of aspartate molecules where (i)

all three carbons, Ca, O and C\ originate from the same source molecule (intact Cr

fragment;/(3)), where (ii) Ca and C^ (f(2)) or (iii) Ca and C (/^ ^) arise from the same

source molecule while the remaining does not (intact C2-fragment), or where (iv) all

three carbon atoms originate from different source molecules (f^) (Fig. 1.3).

The patterns of intact carbon fragments observed in the amino acids represent intact

carbon fragments in eight principal intermediates of central metabolism from which

the amino acids are synthesized, i.e. ribose-5-phosphate, erythrosc-4-phosphate, 3-

phosphoglycerate, phosphoenolpyruvate, pyruvate, acetyl-CoA, 2-oxoglutarate and

oxalacetate. The fractional 13C-labeling approach directly monitors the transformation

of the carbon skeletons of these various intermediates in the metabolic network. The

patterns of intact carbon fragments observed for the different metabolites depend on

the sequence of reactions through which a given molecule has been processed, and the

metabolic origin of an intermediate can therefore be inferred from the presence or

absence of certain intact carbon fragments that are diagnostic for a given pathway. For

instance, oxalacetate molecules possessing intact Co~C} connectivities can only derive

from the anaplerotic pathway (see Fig. 1.4), or phosphoenolpyruvate molecules with

intact Q-C2 connectivities but cleaved C2-C3 connectivities must have been

synthesized from oxalacetate via the gluconeogenic phosphoenolpyruvate

carboxykinase reaction. For a given metabolite, the fraction of molecules that have

been synthesized via a given pathway can be determined from the relative abundance

1.1. The fractional carbon-13 labeling approach for metabolic studies 21

ra(HC)

Jb±L7(U(P to(»C)

12,c=o

Jf=±L

FcPH#èîr#QJ!o ^tM^m

Asp-a

ii

^ ii \1

1,

ftIi

III,

/J l

i

!| H

40

i

0 .40

to(nC)[llz]

Ï Probabilistic equations

rß f(D

if

pa

1C=0

Cß f(2)

1/-<a

1c=o

CP y (2*)

c"

1c=o

CP W3)

1Ca

1c=o

21 % 17% 35 % 27%

Figure 1.3: Determination of the relative abundance of intact carbon fragments

from a single source molecule in aspartate.

(1) Isotopomeis and coitespondmg IlC multiplets for the central caibon atom in the 0-Ca~C

fragment of aspartate. (2) Experimental l3C-l3C scalar coupling tine structrure of Asp-a, from a

preparation with Bacillus subtil is, grown in a minimal medium containing 15 % fully ljC-labeled

glucose and 85 % non-enriched glucose (Sauer et al.. 1997). The intensities of the individual

multiplet components in the nC-nC fine structure are weighted by the relative abundance of the

corresponding isotopomers (1). (3) Relative abundance of intact cat bon fragments (thick lines)

from a single source molecule ('/values', for definitions see also Box 2. h m Asp c"-Ca-C\ in

percent of the total pool, calculated from the relative intensity of the multiplet components

using probabilistic equations (Sz>perski. 1995).

22 Introduction

of such diagnostic intact carbon fragments, and defines a ratio between the fluxes

leading to the observed metabolite pool. This analysis yields information on flux

distribution in several key pathways, such as glycolysis, the pentose phosphate

pathway, the tricarboxylic acid cycle and the Cj-metabolism (Szyperski, 1995; 1998;

Szyperski et al., 1996; 1999). The calculation of a flux ratio is illustrated in Fig. 1.4 for

the example of oxalacetate synthesis in Bacillus subtihs (Sauer et al., 1997). The

existence of a certain pattern of intact carbon fragments may also indicate the presence

of a pathway not recognized originally as part of the metabolic network. This is very

valuable information because the topology of the network may not be determined

under the genetic or environmental conditions of interest. By comparing the patterns of

intact carbon fragments between different metabolites, precursor-product relationships

can be established, for example to explore amino acid biosynthesis pathways (see

chapter 2). Furthermore, when alternative labeling patterns arc generated after a

forward-backward-forward reaction sequence of a metabolite, in some cases the

relative forward and backward rates of a reversible step in the metabolic network can

be estimated.

The fractional C-labeling approach provides information both on active

biosynthetic pathways and on flux ratios in a single experiment, the observed patterns

of intact carbon fragments representing a kind of a 'fingerprint' of the current

regulatory state of the cell. The derived flux ratios can be used to complement

metabolic flux balancing (Sauer et al., 1997; Varma and Palsson, 1994). Metabolic flux

balancing has yielded important information on intracellular flux distributions in many

bacteria (Tsai et al., 1996; Vallino and Stephanopoulos, 1993; Varma and Palsson,

1994). It combines data on uptake and secretion rates, biosynthetic requirements,

metabolic stoichiometry, and quasi-steady state mass balances on metabolic

intermediates, which provides a mathematical framework to calculate intracellular

fluxes. However, since the number of unknown fluxes generally exceeds the number of

intermediates, the resulting equation system is usually underdetermincd and a unique

solution to the llux distribution does normally not exist. Therefore assumptions are

introduced concerning the generation of biosynthetic reducing power (NADPII), energy

stoichiometry, or biological functions of the metabolic system, which might, however,

not hold true under the given experimental conditions. The introduction of additional

1.1. The fractional carbon-13 labeling approach for metabolic studies 23

50%

Anapleroticreaction

(Pyruvate

carboxylase)

/ 4COO" 4COO"

/ 3CH2

^^\ 2c=o

3CH2J2C=0

\ ^OO" ^00"

\ 56% 44%

2-Oxoglutarate

3Ctt»

Jc=o

xcoo

88%

Pyruvate

Oxalacetate

Figure 1.4: Anaplerotic synthesis of oxalacetate: Calculation of a flux ratio from

the relative abundance of intact carbon fragments in the amino acids.

Oxalacetate molecules with intact C-j-Q fragments (bold lines) can only derive from pyruvate

via the anaplerotic pathway (pyruvate carboxylase), and not from 2-oxoglutarate via the

tricarboxylic acid cycle (TCA). This is because all molecules in the 2-oxoglutarate pool exhibit

cleaved C3-C4 connectivities (dotted lines), which are generated when 2-oxoglutarate is formed

by condensation of acetyl-CoA with oxalacetate in the TCA cycle. Intact C2-C3 fragments in

oxalacetate are observed as intact Ca-c'5 fragments in aspartate (compare Fig. 1.3, [f1-2^ + f(3)l

Asp-a). The relative amount of oxalacetate that has been synthesized via the anaplerotic pathway

(50 % = (44 / 88) * 100 rf) is calculated by relating the fraction of oxalacetate molecules with

intact C2-C3 fragments (44 7) to the fraction of molecules with intact Ci-C^ fragments in the

pyruvate pool (88 %; assessed from alanine). In this example, both the anaplerotic flux and the

TCA equally contribute to the synthesis of oxalacetate. Numbers are taken from the analysis of

riboflavin producing Bacillus subtilis (Sauer et al., 1997).

24 Introduction

NMR-derived constraints between intracellular fluxes reduces the uncertainties in flux

estimates from unproven assumptions. For example, a comprehensive flux analysis in

riboflavin-producing Bacillus subtilis illustrated that the NMR-derived constraints are

mandatory for a reliable estimate of intracellular fluxes (Sauer et al., 1996; 1997).

In the metabolic studies presented in the next two chapters, the fractional C-

labeling approach was applied to explore amino acid biosynthesis pathways in the

extremely halophilic archaeon Haloarcida hispanica, and to perform a quantitative

analysis of flux distributions in Escherichia coli central metabolism during exponential

growth in D20-containing minimal media.

25

2. Amino acid biosynthesis in the halophilic

archaeon Haloarcula hispanica

Halophilic archaea are aerobic chemo-organotrophs that grow on a variety of

carbon sources. The central carbon metabolism of some species is relatively well

explored (Danson, 1993; Rawal el al, 1988), while comprehensive investigations of

amino acid metabolism have so far been pursued only for organisms belonging to other

phylogenetic groups (Lodwick et al.. 1991) within the domain of the archaea (Woese

et al., 1990), i.e., various methanogens (FTkmanns et al, 1983; Ekiel et al., 1984; Ekiel

et al., 1985a; 1985b; Sprott et al., 1993) and the anaerobic, extremely thermophilic

Thermoproteus neutrophilia (Schäfer et al., 1989). These studies indicated that most

amino acids in thermophilic and methanogenic archaea are synthesized via pathways

that had previously been described for bacteria and eukarya (Gottschalk, 1986;

Neidhardt et al., 1996; Vogel, 1960). An extension of such studies to halophilic archaea

is thus of interest for obtaining new insights into the evolution of carbon metabolism in

general. Furthermore, organisms living at extreme environmental conditions

(extremophiles) are gaining increasing importance for biotechnological applications

(Davis, 1998), and the analysis of their metabolism constitutes a prerequisite foi-

possible future metabolic engineering (Bailey, 1991).

The present investigation of the amino acid biosynthesis in the halophilic archaeon

Haloarcula hispanica was initiated because of the potential biotechnology interest.

H. hispanica can efficiently use glycerol for amino acid synthesis (Juez et al., 1986).

We primarily employed biosynthetically-directed fractional C-labeling (Sauer et al.,

1997; Senn et al., 1989; Szyperski, 1995; 1998; Szyperski et al., 1992; 1996; 1999;

Wüthrich et al, 1992) with glycerol as the sole carbon source.

1 ^Fractional C-labeling was achieved by growing H. hispanica in a minimal

medium containing approximately 10% uniformly 13C-labeled glycerol and 90%

glycerol containing C at natural abundance. Cells were grown in a batch culture and

harvested in the mid-exponential and the early stationary phase in order to assess

possible changes of the metabolic state during growth. After harvest, the cells were

26 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

lysed by osmotic shock. The cytosolic proteins were precipitated with ethanol and

subsequently hydrolysed in hydrochloric acid (Box 2.1). The 2D [ljC,1Hl-HSQC

spectra were analysed as described by Szyperski (1995) (Table 5.1, appendix). The

observed relative multiplet intensities were used to calculate the relative abundances of

intact carbon fragments (Szyperski, 1995) (Box 2.1).

Box 2.1: Experimental methods

Growth of the organism and sample preparation. Following the protocol developed by

Rangaswamy and Altekar, 1994, H. hispanica (Juez et al, 1986) was grown in a medium

containing per liter 200 g of NaCl, 36 g of MgS04 7H0O, 6 g of Tris base, 4 g of KCL 1 g of

CaCl0 H20, 2 ml of FeS04 711,0 (0.4% in 1 mM HCl). 2 ml of K9HP04 (5% in distilled water)

and 5 ml of NH.CT (20% in distilled water). Glycerol (20 ml, 25% in water) was added, and the

pH was adjusted to 7.5 with HCl prior to sterilization (filter pore size cut-off 0.45 um). In the

standard experiments, 10% of the glycerol was uniformly l3C-laheled. In the experiment with

C-labeled threonine, no labeled glycerol was used and 307 mg of (13C4]~threonine per liter was

added under otherwise identical conditions. Cells were grown in six 35 ml cultures that were

shaken at 100 rpm in 100 ml Erlenmeyer flasks for 7 days at 40°C until the early stationary phase

was reached. Cells from cultures in the mid-exponential growth phase were harvested after 3 days.

After centrifugation at 3'000 x g, the cells were taken up in 10 ml of water and frozen in liquid

nitrogen. No DNase was added in order to prevent contamination of the halohacterial proteins with

unlabeled amino acids. After being warmed to 0°C. the slurry was centrifuged at 20'000 x g, and

the protein in the clear supernatant was precipitated with 65% ethanol at a temperature of -20T

over night. After centrifugation, the pellet was lyophilized and hydrolyzed after addition of 3 ml of

6 M HCl at 80°C for 2 days in a sealed Pyrex tube, yielding about 70 mg of dried biomass.

NMR spectroscopy. NMR experiments were performed at 40°C. Proton-detected 2D l!3C,'H]-heteronuclear single-quantum correlation spectra were recorded with the pulse sequence devised

by Bodenhausen and Ruben (1980). which ensures that 'H-i3C scalar couplings do not affect the

3C- C scalar coupling fine structure along (o^'^C) (Fig. 1 in Szyperski et al.. 1992). Pulsed field

gradients were employed for coherence pathway rejection (Bax and Pochapsky, 1992; Wider and

Wüthrich, 1993), and a 2 ms spin-lock pulse (Otting and Wüthrich. 1988) was used to purge the

magnetization arising from '"C-bound protons and the residual 2HOH signal. 13C-decouplingduring t2 was achieved with the composite pulse decoupling scheme GARP (Shaka et al, 1985),

and quadrature detection in co, was accomplished with States-TPPl (Marion et al, 1989). For the

samples obtained from the cultures grown with Ll3C3"|-glycerol, two spectra were recorded, i.e.,

one focused on the aliphatic carbons, with the' 3C-carrier set to 42.5 ppm relative to the chemical

shift of 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS), and one for the aromatic rings,

with the C-carrier set to 125.9 ppm.

Box 2.1: (continued)

NMR spectroscopy (continued). For the sample obtained from the culture grown with

L C41 -threonine, only the spectrum focussing on the aliphatic carbons was recorded. The spectra

for the aliphatic resonances were folded along C0|(13C), with a sweep width of 33.8 ppm.

Somewhat different experimental conditions were chosen for the individual measurements. For the

sample harvested during the mid-exponential growth phase and the sample generated with

I C4]-threonine, the aliphatic spectra were recorded at a C resonance frequency of 125.8 MHz,

with a Bruker DRX500 spectrometer. The measurement time was 9 h per spectrum (F706 x 256

complex points; maximal evolution times ?|max = 402 ms; t2mdX = 102 ms, relaxation delay

between scans 2 s). For the sample harvested in the early stationary growth phase, the aliphatic

spectrum was recorded in 8 h at 188.6 MHz, with a Bruker DRX750 spectrometer (2'559 x 512

complex points; î]max = 392 ms; t2mäx = 87 ms, relaxation delay between scans 2.4 s). The

aromatic spectra were recorded in 3 h, with a Bruker DRX500 spectrometer (920 x 512 complex

points; tïmixx = 392 ms; t2mdX = 87 ms, relaxation delay between scans 1 s). The data were

processed with the program PROSA (Güntert et al, 1992). Before Fourier transformation, the time

domain data were multiplied in t\ and t2 with sine-bell windows shifted by n/2 (DeMarco and

Wüthrich, 1976). The digital resolution after zero-filling was 1.0 Hz/point along töt and

2.4 Hz/point along (02 for the aliphatic spectra at 125.8 MHz, and 0.6 Hz/point along (ol and

5.8 Hz/point along (o2 for the aromatics. For the 188.6 MHz spectrum the digital resolution was

0.8 Hz/point along co, and 3.7 Hz/point along (ù2.

For the experiments using [ C^l-glycerol as the LC source, the overall degree of C-labeling in

the amino acids, denoted as p| in the probabilistic equations of Szyperski ( 1995), was determined

from the satellites of selected well-separated peaks in ID !H NMR spectra (tmAX = 1.022 s,

relaxation delay between scans 10 s). p] was 0.127 for both preparations used here.

Data analysis. The individual multiplet components of the I3C-13C scalar coupling fine structures

were integrated using the program XEASY (Bartels et al., 1995) and FCAL (Glaser, 1999), and

the observed relative multiplet intensities were used to calculate the relative abundances of intact

carbon fragments (Szyperski. 1995). Following the definitions by Szyperski, (1995) and

Szyperski et al. (1996). f'1-1 represents the fraction of molecules in which the observed carbon

atom and its neighbouring carbons originate from different source molecules of glycerol, and

/^ the fraction of molecules in which the observed carbon atom and at least one neighbouring

carbon originate from the same source molecule. For a central carbon in a C3 fragment that

exhibits different 13C-13C scalar coupling constants with the two attached carbons, f(2)

represents the fraction of molecules for which the central carbon and the carbon with the

smaller coupling come from the same source molecule, while f (2") is used if the carbon with

the larger coupling comes from the same source molecule as the observed carbon. /'(3^ denotesthe fraction of molecules in which the observed carbon atom and both neighbours in the C3-

fragment originate from the same glycerol molecule (Fig. 1.3).

28 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

Virtually identical C scalar coupling fine structures were observed for samples

taken from the mid-exponential growth phase and the early stationary phase, and hence

there are no significant differences in the resulting relative abundances of intact carbon

fragments, as collected in Table 5.1 (appendix). This suggests that there are no major

differences in flux ratios through the central metabolic network when the two growth

periods are compared (Szypcrski, 1995; 1998; Szyperski et al.. 1992; 1996; 1999).

During hydrolysis, cysteine and tryptophan were oxidized and could thus not be

evaluated, and asparagine and glutamine were deamidated to aspartate and glutamate

(Szyperski et al., 1992; Wüthrich et al., 1992). The ring carbons of phenylalanine were

not evaluated because of strong-coupling effects (Szyperski, 1995). The evaluation of

the observations for all other carbon positions (Table 5.1, appendix) showed that

except for isoleucine, lysine and the aromatic ring of tyrosine, the proteinogenic amino

acids in H. hispanica are synthesized according to the pathways commonly found in

both bacteria and eukarya (Gottschalk, 1986; Neidhardt et al., 1996; Umbarger, 1978;

Voet and Voet, 1995).

Amino acid synthesis from glycolytic intermediates. Identical values for intact

carbon fragments (/'values; for the definitions see Box 2.1 and Fig. 1.3) are observed

for phenylalanine and tyrosine, where /(3){Phe-a) approximately equals/^{Tyr-a}which approximately equals 1 (Table 5.1, appendix). This finding is in agreement with

the shikimate/chorismate pathway (Bentley, 1990; Umbarger, 1978, Voet and Voet,

1995), where the cP-Ca-C fragments of phenylalanine and tyrosine are both derived

from phosphoenolpyruvate, which is itself expected to derive from glycerol without

cleavage of carbon-carbon bonds (Rawal et al., 1988). Serine appears to be

synthesized from 3-phosphoglyceratc, which is also directly derived from glycerol.

However, reversible interconversion into glycine and a C{ unit leads to cleavage of

CP-Ca connectivities, so that /' (1){Ser-ß} approximately equals 0.55 in both

preparations (Table 5.1, appendix). Moreover, the fact that [/'(2') + / (3)]{Ser-a}

approximately equals/^(Gly-a) (Table 5.1, appendix) shows that glycine is nearly

exclusively derived from Ca-C of serine.

Virtually identical / values are detected for Val-yj, Leu-Sj and Ala-ß, which

provides evidence that valine, leucine and alanine are derived from pyruvate according

to the well-known biosynthetic pathways (Umbarger, 1978, Voet and Voet, 1995). This

29

is further supported by the following observations: [f( )+ f( j]{Ala-a} ~

[/•(2,)+/(3)]{Val-a}, and/'(1){Leu-ß} «/(1){Val-Y2H f(1){Leu-S2} - 1 (Table 5.1)

(Szyperski, 1995). That approximately equals f ^{Ala-ß} ~ /(2'^(Leu-a} further

shows that Leu-a is derived from C2 of acetyl-CoA.

The /values of His-a, His-ß and His-0 (Table 5.1, appendix) show that the

precursor for histidine, ribose-5-phosphate. is synthesized from glyceraldchyde-3-

phosphate and fructose-6-phosphate via the non-oxidative part of the pentose

phosphate pathway (Fig. 2.1). The presence of transketolase (U.C. 2.2.1.1) and

transaldolase (E.C. 2.2.1.2) has been reported for other halophilic archaea (Danson,

1993; Rawal et al., 1988). The equations f(3){His~a] =/"(3){Phe-a} =/(3){Tyr-a} - I

and/'(2){His-ô} -/'^{Phe-ß} «/"^{Tyr-ß} ~ 1 indicate that histidine is composed of

a C^-Ca-C fragment and a C -CY fragment from two different glycerol molecules

(Fig. 2.1). This is due to the facts that (i) the glyeeraldehyde-3-phosphate pool is almost

exclusively derived from glycerol without cleavage of carbon-carbon bonds (see

above), and that (ii) fructose-6-phosphate is synthesized from two glycerol molecules

via reversal of glycolytic reactions (Danson, 1993; Rawal et al., 1988), so that it

comprises the intact fragments Cj-CV-C:. and C^-C^-Cg from two different glycerol

molecules.

The / values that characterize pyruvate and phosphoenolpyruvate are slightly

different, i.e., pyruvate exhibits about 5 % cleavage of C3-C2 connectivities, as

evidenced by the equation f(1){Ala-ß} =/'(1){Val-Y[} ~/(l){Leu-8i} =/a){lle-Y2} ~

0.05, whereas only intact C3-C2 connectivities are detected for phosphoenolpyruvate

(/^{Phe-cc} ~ /'^{Tyr-a} ~ 1) (Table 5.1, appendix). This suggests that, in addition

to the synthesis of pyruvate from phosphoenolpyruvate via pyruvate kinase (E.C.

2.7.1.40) (Rawal et al., 1988). the malic enzyme (E.C. 1.1.1.38, 1.1.1.39, and 1.1.1.40)

contributes to pyruvate synthesis via oxalacetate. In fact, malic enzyme activity has

been reported for other archaea, such as Halobaciermm salinarium (Bhaumik et al.,

1994), Halobacterutin cuiirubrum (Cazzulo and Vidal. 1972) and Sulfolobus

solfataricus (Bartolucci el al., 1987).

30 Ammo acid biosynthesis m the halophilic archaeon Haloarcula hispanica

CH,OHI

"

c=o

ClbOPO-r CTbOII1 IC=0 ^_ ClbOIlI I

-

CH2OH CIl2OH

dihydroxyacetone-phosphate glyeei ol

HO-CI I

I1C-OH ÇHOHC-OH HÇ-OHCH2OPO,2~ CH2OPO,2-

f i uctose-6-phosphate glyceraldehydc- ^-phosphate

CHoOH

ÏCHO C=0

HC-OH HO-CH -*--*- histidine

1 IHC-OH IIC-OH

CH2OPO32 ch2opo>

eiythrose-4-phosphate \\ lulose-5-phosphate

Figure 2.1: Intact carbon fragments in intermediates of the pentose phosphate

pathway.

Synthesis of erythrose-4-phosphate and xylulose-5-phosphate from glycerol via reversal of

glycolytic reactions and the action of transketolase (EC. 2.2.1.1). Thick lines indicate

carbon-carbon connectivities arising from a single source molecule of glycerol (see the text).

Amino acid synthesis from intermediates of the tricarboxylic acid cycle. The

/values observed for Asp-a and Asp-ß coincide with those observed for Thr-a and

Met-a (Asp-a), and Thr-ß (Asp-ß). Moreover, virtually identical/values were found

for Glu-a and Pro-a, for Glu-ß, Pro-ß and Arg-ß; for Glu-y and Pro-y; and for Pro-5

and Arg-6 (Table 5.1, appendix). This is consistent with the well known pathways in

which aspartate, threonine, and methionine are derived from oxalacetate, and in which

2-oxoglutarate serves for the biosynthesis of glutamine, proline, and arginine

(Szyperski, 1995). Furthermore, the following observations demonstrate that

2-oxoglutarate is formed by irreversible condensation of oxalacetate with acetyl-CoA

31

in the citric acid cycle (Szyperski, 1995): (i) the equation/( qGlu-ß} ~/( ' {Pro-ß} ~

/(2){Glu-y} -/^{Glu-y} -/^{Pro-y} ~ 0 shows that intact C3-C4 connectivities in

2-oxoglutarate are absent, (ii) the f-values observed for Asp-ß (and also Thr-ß) are

equal to those observed for Glu-cx (and Pro-a), which is in agreement with the fact that

the C1-C2-C3 segment of 2-oxoglutarate is derived from C2-C3-C4 of oxalacetate,

and (iii) the equation f{l ^{ Glu-y} ~ f(2 ^Feu-a} =/^2){Pro-8} ~/(2^{Arg-8} shows

that C4-C5 of 2-oxoglutarate is derived from acetyl-CoA (Table 5.1, appendix).

32 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

2.1. Isoleucine biosynthesis

A variety of pathways are known for isoleucine biosynthesis in microorganisms.

Most common is the 'threonine pathway', which uses threonine and pyruvate as

precursors (Abelson, 1954; Umbarger, 1978; Voet and Voet, 1995) (Figs. 2.2, 2.3). In

the 'pyruvate pathway', isoleucine biosynthesis proceeds from pyruvate and

acetyl-CoA (Charon et al., 1974) (Figs. 2.2, 2.3), whereas in the 'glutamate pathway',

glutamate and pyruvate serve as precursors (LeMaster et al., 1982; Phillips et al., 1972)

(Figs. 2.2, 2.3). In rare cases, isoleucine synthesis has also been found to proceed from

homoserine (Flavin and Segal, 1964; Vollbrecht, 1974), propionate (Ekiel et al., 1984;

Monticello et al.. 1984; Sauer et al., 1975). or 2-methylbutyrate (Ekiel et al., 1984;

Monticello et al., 1984; Robinson and Allison, 1969). In all pathways except the one

starting from 2-methylbutyrate, a-ketobutyrate serves as an intermediate, and pyruvate

yields the CY -C^ fragment of isoleucine.

A

GLHlHlHi] /^tt^W^VAt^ CD-O-OD ©-CDoxalacctaie/thi eomnc 2-oxoglmai ate pyiuvute acetyl-CoA

B

isoleucine via pyruvate pathway via tliteomne pathway via glutamate pathway

5 Yi 5 yi 8 Yi

(D—(DvP a4-3. P a

^D—©—(T)"

J2y- 2 -

A"7£V ß a

Y2y? li

Figure 2.2: Precursors for isoleucine biosynthesis.

Incorporation of oxalacetate or threonine. 2-oxoglutarate. pyruvate and acetyl-CoA, for which

carbon skeletons are schematically shown in (A), into the carbon skeletons of isoleucine (B).

according to the biosynthetic pathways shown in Fig. 2.3. Note that threonine is synthesized from

oxalacetate without rearrangement of the carbon skeleton (see the text). The notation of the

carbon atoms follows IUPAC-1UB recommendations (1LJPAC-IUB commission, 1970), i.e.,

02C(4)-C(3)H3-C(2)0-(1)03- for oxalacetate, -02C(5VC(4)H2-C(3)H2-C(2)O^C(l)02- for

2-oxoglutarate. C(3)H3-C(2)0-C(1)CV for pyruvate, and C(2)H3-C(l)0-SCoA for

acetyl-CoA.

2.1. Isoleucine biosynthesis 33

SCoAIC=0

ICFb

AcetvI-CoA

COO-Ic=o

ICH,

Pyruvate

COO"

ICH,

I-

HO—C-CH-,

coo

Citiamalate

COO"

H^N-CHI

HC-OH

ICH,

Threonine

coo-

+H-,N-CH

CH-,

I"

CH,

I"

COO

Glutamate

3 t

COO-

%N~CHI

HC-CH,

COO

ßAlethylaspaitatc

x-Ketobut\rate k

COO

c=o

IHC- CH,

CH;

CH,

a-Keto-ß-meth} K alci ate

coo-

I- c=o

CH,

Pyruvate

COO

IfH,N-Clt

IHC-CH,

I

CH2

CH3

isoleucine

Figure 2.3: Three routes for isoleucine biosynthesis.

Pyruvate and acetyl-CoA are the precursors for isoleucine synthesis via the "pyruvate pathway'

(route 1) (Charon et al.. 1974). threonine and pyruvate are the precursors for isoleucine synthesis

via the 'threonine pathway" (route 2) (Abelson, 1954; I Imbatger. 1978; Voet and Voet, 1995), and

glutamate and pyruvate are the precursors for isoleucine synthesis via the "glutamate pathway'

(route 3) (LeMaster and Cronan. 1982; Phillips et al.. 1972). a-ketobutyrate is a common

intermediate in these three pathways.

34 Amino acid biosynthesis m the halophilic aichaeon Haloarcula hispanica

In H. hispanica, Ile-y2 exhibits the same disttibution of intact carbon fragments

originating from a single molecule of glycerol as Ala-ß (Table 5.1, appendix),

indicating that the CY -Cr fragment of isoleucine and the C^-Ca fragment of alanine

are both derived from pyruvate (Fig. 2.2, Fig. 2.3). Ho\ve\er, the relative abundances

of intact carbon fragments determined at Ile-a and Ile-5 cannot be explained by a

single one of the possible individual pathways (Table 2.1, Box 2.2). A satisfactory fit

of the data was obtained with the assumption that the threonine and pyruvate pathways

operate simultaneously in a split-pathway tashion Foi the early stationaiy growth

phase, the decomposition ot the 13C-[ C fine structures at both lle-a and Ile-5

indicates that 56% of isoleucine is synthesized via the threonine pathway and 44% via

the pyruvate pathway, and virtually identical \ alues were obtained for the mid-

exponential phase (Table 2 1, Fig. 2 4)

Table 2.1: Exploration of isoleucine biosynthesis

Relative abundance 01I

caibon atom

indicated intact caibon fragment" at

Pathwav(s) Mid-exponential Fatly stationaiyanalysed phiise

2)0l/(2) "7i>"phase

f(\) /( f7)0lf(2)

Expetimental'7 He 8b 051 0 69 0 32 0 68

Ile a 0 16 0 84 0 17 0 83

Pymvate and 1 hieoninef He 8b 051 0 69 0 32 0 68

lie a 0 16 0 84 0 17 0 85

Pyruvate He sb 0 05 0 95 0 05 0 95

(piecursois, pymvate and <ice tyl CoV) Ile-a 0 05 0 95 0 05 0 95

Threonine' lle-o* 051 0 49 0 55 0 47

(piecuisots. thieonme and p\ i in ate) Ile a 025 0 75 0 26 0 74

Glutamate^ Tie 8h 100 0 00 0 98 0 02

(piecuisois, glutamate and P1* i in ate) Ile a 0 56 0 44 0 57 0 43

"Foi the dclmition ol /' " /( '

and f{ 'see Box 2 1 and 1 is 1 5 /,-1 denotes tiactions foi lie <5 f(1 )

denotes hachons foi

Ile a /(7,{lle a) =/n){Ilc a} = 0 00 (see Table 5 1 Tis 2 2) <\ll values except the expenmental values aie piedictions''The ft actions foi Ile yl aie not gnen since those duned fiom lie 5 and He Yi piOMde the same mfotmation

(S/ypetski 1W5)'

Contiîbutions of the pvnnatc and thieonme pathway 44 and 56 cc îespeclnelyll

Calculated with equations 2 2 1 and 2 2 2 (Box 2 2)' Calculated with equations 2 2 5 and 2 2 4 iBox 2 2)1 Calculated with equations 2 2 5 and 2 2 6 i Bo\ 2 2)

2.1. Isoleucine biosynthesis 35

threonine pathway pyruvate pathway 13C-flne structure

0.56 x Ile-a

JUL I I

0.56 x lle-ô

0.44 x Ile-a

J !

0.44 x lle-0

1

lle-a

t0(15C)26 6 26 4[ppml

lle-ô

47 7 47 5

Figure 2.4: Contributions of the threonine and pyruvate pathways to isoleucine

biosynthesis.

Decomposition of the experimental C- C scalar coupling fine structures for the Ile-a and Ile-ö

carbons into contributions from the threonine and pyruvate pathways (Fig. 2.3, Table 2.1). The

stick diagrams represent the fine structures that would be expected if only a single pathway were

operational, and the experimental cross sections on the right were taken along cot( C) from the

2D [13C,JH1-ITSQC spectrum recorded with biomass that was harvested in the early stationary

phase (see the text). 56 % and 44 % of isoleucine are synthesized via the threonine and pyruvate

pathways, respectively. The carbon chemical shifts are relative to those of DSS (2,2-dimethyl-2-

silapentane-5-sulfonate sodium salt).

To verify that the threonine pathway, which has so far not been described for

archaea, is operational in H. hispanica, we performed an additional labeling

experiment using [ C4]-threonine instead of [13C3]-glycerol (see Box 2.1). Consistent

with the presence of the threonine pathway, the C- C scalar coupling fine structures

observed at Ile-8, Ile-Yp and lle-a are dominated by a doublet (Fig. 2.5), which proves

that intact Ç°-Cy[ and Ca-C fragments originate from [ LlC4|-threonine (Fig. 2.2). The

poor signal-to-noisc ratio of the y2-carbon cross peaks indicates that this carbon

position is only sparsely enriched with 13C. Consistently, the y2-carbon exhibits a fine

structure that is also observed at Ala-ß. This is due to the fact that the Cy2-C^ fragment

36 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

is derived from pyruvate in both the threonine and the pyruvate pathways (note that

pyruvate serves for alanine biosynthesis). Long-range"

Tcac5 couplings of 3.1 Hz are

observed as an additional splittings of the doublet lines at Ile-a and Ile-o, and jJcy2C'

couplings of 1.7 Hz (see Krivdin and Delia, 1991) in threonine are observed as

broadenings of the doublet lines at Thr-Vi (Fig. 2.5). The observation of these long-

range carbon-carbon couplings confirms that both the C -CYl and Ca-C fragments in

isoleucine are derived from the same single threonine molecule via a-ketobutyrate.

Alternative combinations of multiple biosynthesis pathways are not supported by

the experimental data. For example, decomposition of the isoleucine C- C fine

structures into fractions stemming from the pyruvate and glutamate pathways leads to

different contributions for the two pathways when they are derived either from Ile-a or

from lle-ô, both for the mid-exponential and the early stationary phases (Table 2.1).

Decomposition into the threonine and the glutamate pathways yields negative

contributions for one of the pathways. Finally, if one assumes simultaneous operation

of all three pathways of Fig. 2.3. one finds that the contribution of the glutamate

pathway would be below 10 %, while the relative contributions of the pyruvate and

threonine pathways would be similar to those obtained when only those two pathways

are assumed to be active (see above and Fig. 2.4).

2.1. Isoleucine biosynthesis 37

(A) Thr-a

„(13C) 27 8 27 3 [ppm]

(D) lle-a

i

26!

(B) Thr-Y2

t

55.8 55 4

26 3

(E) lle-ô

47 8 47 4

(C) Ala-ß

52.0 51.6

(F) lie-y

51.0 50 6

Figure 2.5: Evidence of the existence of the threonine pathway from a labeling

experiment with [ C^-threonine.

Cross sections taken along co(( C) from the 2D [LC.'ll|-HSQC spectrum recorded with the

biomass labeled with L^CaTthreonine. (A to C) LC-nC scalar coupling fine structures of the

precursors used for isoleucine biosynthesis via the "threonine pathway' (Fig. 2.3). i.e., Thr-a.

Thr-Yo and Ala-ß representing C^ of pyruvate. (D)-(F)l V-' C fine structures observed at lle-a,

lle-ô and Ile-Y2 01e~Yi yields the same information as Ile-5 [Szypcrski. 1995]). The fine structures

of Ile-a and Ile-5 are dominated by a doublet, which proves that the fragments C'°-CY and

Ca~C originate from [ 13C4Fthi*eonine. The Yo-carbon exhibits a] JC-A C fine structure similar to

that of Ala-ß, which is consistent with the fact that the CY~-0 fragments of isoleucine are

derived from pyruvate. The additional small splittings of the doublet components of lle-a and

Ile-5 (D and E). and the broadening of the Thr-Y2 doublet lines (B) arise from the vicinal scalar

couplings JrjaCS m isoleucine and ~Jçy2c in threonine (Krivdin and Delia. 1991) (see the text).

The carbon chemical shifts are relathe to DSS. The asterisk in panel E indicates an impurity.

38 Annuo acid biosynthesis in the halophilic archaeon Haloarcula hispanica

Box 2.2: Calculation of the relative abundances of intact carbon fragments in

isoleucine that are expected for specific biosynthetic pathways.

The relative abundances of intact carbon fragments that are expected for specific biosynthesis

pathways can be calculated from the relative abundances found in the respective precursors (Fig.

2.2). C^-C^ of He derives from pyruvate in all pathways considered (Figs. 2.2, 2.3), and Ile-Y2 is

therefore not used in the calculations. For the definitions of/values see Box 2.1 and Fig. 1.3.

(a) Pyruvate pathway. Pyruvate and acetyl-CoA are assessed via Ala-ß and Leu-a, respectively.

/(1){Ile-5} =/(n{ Ala-ß}, /(2){Ile~5} = f(2)| Ala-ß} (2.2.1)

/(l){Ue-a} = [f(1) +/(2)){Leu-a}, /^{Ue-a} = lf(2,) + f(3)]{Leu-a} (2.2.2)

(b) Threonine pathway. Threonine is directly assessed.

/(l){lle-5} =/(1){Thr-Y2}. /(2){Ile-5} =/(2){Thr-Y2} (2.2.3)

/(l){Ile-a} = [f{l) +/(2)]{Thr-a}, /(2<){He-a} = [/(2,) +/(3)l{Thr-a} (2.2.4)

(c) Glutamate pathway. Glutamate is directly assessed.

/(1){Ile-5} = [f(l) +/(2n]{Glu-Y}, /(2){He-ô} = [f(2) +/(3)1{G1u-y} (2.2.5)

/(l){IIe-a} = [f(]} +/{2)]{Glu-a), ,f(r }{IIe-a} = l/'(2<) + f(3)l{Glu-a) (2.2.6)

2.2. Lysine biosynthesis 39

2.2. Lysine biosynthesis

Lysine biosynthesis via the 'diaminopimelate pathway' starts from aspartate and

pyruvate, whereas the 'a-aminoadipate pathway' relies on 2-oxoglutarate and

acetyl-CoA as precursors (Bhattacharjee, 1985; Schäfer et al, 1989) (Figs. 2.6. 2.7).

Two variants of the diaminopimelate pathway differ in the reaction sequence used to

convert L-À -piperidme-2.6-dicarboxylate to D,L-diaminopimelate (Fig. 2.6B). In the

dehydrogenase variant, L-A -pipendine-2,6-diearboxylate is directly converted to

D,L-diaminopimelate by diaminopimelate dehydrogenase (EC 1.4.1.16) (Misono and

Soda, 1980; Misono et al., 1979; White, 1983). In the acetylase/succinylase variant

(Berges et al., 1986; Kindler and Gilvarg, 1960; Sundharadas and Gilvarg, 1967), I -A1-

piperidine-2,6-dicarboxylate is first acetylated or succinylated and then converted to

the symmetric intermediate L,L-diaminopimelate, which is finally epimeri/ed to

D.L-diaminopimelate. The dehydrogenase variant can readily be distinguished from the

acetylase/succinylase variants in the fractional labeling experiment, because the

involvement of the symmetric intermediate L.L-diaminopimelate implies

symmetrization of the 13C-labeling pattern about the C^-CY bond (Fig. 2.7).

40 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

Bo

IIcirf-c— scoa

Acetyl-CoA

o

ooc—ni—chu—au—coo

oKetoglutarate

Nil-,'"I

ooc—en— cn2—coo

L-Aspartate

' !"OOC—C —OH, -~^ t

V\ rin ate

ooc COO

L-A -Pipeiidmc-2.6-dicarboxylale

NI1,+I

-ooc—en—cn2— cn2—cu2— coo

a-Aminoadipic acid

1

\

\

,., \ooc—cn—icii-,',—cn—coo-

L L-Diaminopimclatc

I -Glutamate

Saccharopine

n-Kctoelutaialc

Nil,1-

OOC- CH— (CH,),—CH2—MI,""

L-Lysiee

MiC NH3'I I

OOC— CI 1— (CII2)3—CH—COO

n, L-Diaminopime I ale

NH,1"I

OOC—ai~(Cll2)3~CH2-NH,+

Q-O

L-Lysine

Figure 2.6: Lysine biosynthesis pathways.

(A) Synthesis via the a-aminoadipate pathway (Bhattachar)ce. 1985; Schäfer et al., 1989). (B)

Synthesis via the diaminopimelate pathway. In the "acetylase-Asuccinylase variant'. L-A -

piperidme-2,6-dicarboxylate is converted to D.L-diaminopimelate via the symmetric intermediate

L.L-diaminopimelate (route 1) (Berges et al. 1986; Kindler and Gilvarg. I960; Sundharadas and

Gilvarg, 1967). In the "dehydrogenase variant'. L-A -piperidine-2,6-dicarboxylate is directly

converted to D.L-diaminopimelate by diaminopimelate dehydrogenase (route 2) (Misono and

Soda, 1980; Misono et al.. 1979; White. 1983).

2.2. Lysine biosynthesis 41

A

EHIHIHi] A^wiVz^yA CEKiMD ©-Ooxalacetate 2-oxoglutaiate pymvate acetyl-CoA

B

lysine i la L L-diaminopimelale i ta diammopimclate-dehydiogenase

C S Y P «

(I>K3HTHIHIH3 50%

t S y ß a

(^2)-hA)— 4 -JAT"- 2 — 1

c 5 Y ß "

lIlKIHIhCIKIMD 50% i w u-aminoadipate

f 5 Y ß «

Figure 2.7: Precursors for lysine biosynthesis.

Incorporation of oxalacetate. 2-oxoglutarate. pyruvate and acetyl-CoA, for which carbon

skeletons are schematically shown in (A), into the carbon skeletons of lysine (B). according to the

biosynthetic pathways shown in Fig. 2.6. The notation of the carbon atoms follows IUPAC-IUB

recommendations (KJPAC-fUB commission. 1970) (see Fig 2.2).

For H. hispanica our study indicates that biosynthesis during the mid-exponential

as well as the early stationary growth phase proceeds via the dehydrogenase variant of

the diaminopimelate pathway (Figs. 2.6B, 2.7, Table 2.2, Box 2.3). The relative

abundances of intact carbon fragments observed at Lys-5 and Lys-e correspond to the

values observed at Ala-ß (Table 5.1. appendix), which is expected if the fragment

Cc-C8 comes entirely from the fragment C3-C2 of pyruvate when lysine is synthesized

via diaminopimelate dehydrogenase (Fig. 2.7, Box 2.3). The data in Table 2.2 show

that there are no significant contributions to lysine biosynthesis either from the

acetylase/succinylase variant of the diaminopimelate pathway or from the

a-aminoadipate pathway. This result is of special interest since simultaneous operation

of both variants of the diaminopimelate pathway has previously been documented for

the bacterium Corynebacterium glutamicum (Ishino et al., 1984; Schrumpf et al,

1991).

42 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

Table 2.2: Exploration of lysine biosynthesis

Relative abundance of

indicated intact carbon fragment" at:

Pathway carbon atom

analysed

Mid-exponential

phase

/" (2) y(3)

Early stationary

phase

f(1) f(2) /(3)

Experimental

Diaminopimelate-dchydrogenase variant

of the diaminopimelate pathway'

Acetylase/succinylase variant

of the diaminopimelate pathway'

a-Aminoadipate''

Lys-e" 0.04 0.96 0.05 0.95

Lys-ß 0.15 0.78 0.07 0.18 0.77 0.05

Lys-e 0.05 0.95 0.05 0.95

Lys-ß 0.16 0.75 0.09 0.18 0.74 0.08

Lvs-e 0.29 0.71 0.30 0.70

Lys-ß 0.11 0.85 0.04 0.12 0.84 0.04

Lvs-e 0.04 0.96 0.04 0.96

Lys-ß 0.52 0.48 0.00 0.55 0.45 0.00

"For the definitions of/ \ f~\ and /

L 'see Box 2.1 and Fig. 1.3. All \ allies except the experimental values arc expected.

Values coincide with those observed for Ala-ß.'" Calculated with equations 2.3.1 and 2 3.2 (Box 2.3)."

Calculated with equations 2.3.3 and 2.3.4 (Box 2.3).e

Calculated with equations 2,3.5 and 2.3.6 (Box 2.3).

2.2. Lysine biosynthesis 43

Box 2.3: Calculation of the relative abundances of intact carbon fragments in lysine

that are expected for specific biosynthetic pathways.

The relative abundances of intact carbon fragments that are expected for specific biosynthesis

pathways can be calculated from the relative abundances found in the respective precursors (Fig.

2.7). For the definitions of/values see Box 2.1 and Fig. 1.3.

(a) Diaminopimelate pathways. Pyruvate and aspartate are assessed via Ala-ß and Asp-a or Asp-ß,

respectively.

(a.l) Diaminopimelate-clehydrogenase variant

/(|){Lys-e} =/(l){ Ala-ß}. fi2){ Lys-e} =f(2){ Ala-ß} (2.3.1)

/(1){Lys-ß} =/d>{ Asp-ß}, /(2){Lys-ß} = \f(2) +f^][ Asp-ß},

/(3){Lys-ß} =/(3){ Asp-ß} (2.3.2)

(a.2) Acetylase/succinylase variant

/(1){Lys-e} =0.5 (/(1){Ala-ß} + [/'(1)+/(2")}{Asp-a}),/(2){Lys-e} =0.5 (/(2){Ala-ß} + lf(2)+/(3)l{Asp-a}) (2.3.3)

/(1){Lys-ß} = 0.5 (/-(1){Ala-ß} +/'(l){Asp-ß}),

/(2){Lys-ß} = 0.5 (fi2){Ala-ß} + \fi2) +/£*>]{Asp-ß})./(3){Lys-ß} =0.5/(3){ Asp-ß} (2.3.4)

(b) a-Aminoadipate pathway. 2-üxoglutarate is assessed via Glu-y and Glu-a.

/(l){Lys-8} = l/"(1) +/(2)l{Glu-Yh /(2){Lys-e} = [f^ +/(3)]{G1u-y} (2.3.5)

/(1){Lys-ß} = l/,(1) +/(2^]{Glu-a}, /(2){Lys-ß} = }/'(2) +/(3)J{Glu-a}./(3){Lys-ß} = ().(X) (2.3.6)

44 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

2.3. Tyrosine biosynthesis

Commonly, erythrose-4-phosphate and phosphoenolpyruvate serve for the

biosynthesis of the aromatic ring of tyrosine via the shikimate pathway (Bentley, 1990;

Umbarger, 1978; Voet and Voet. 1995). To evaluate the 13C-labeling pattern of the

tyrosine ring in light of this pathway, /values for this pathway were predicted with the

assumption that histidine biosynthesis proceeds via ribose-5-phosphate (Umbarger,

1978; Voet and Yoet, 1995) (Box 2.4). The pentose-pool must then be composed of

molecules carrying intact C1-C2-C\ and C4-C5 fragments (Fig. 2.1; see above, /values

obtained for His), so that erythrose-4-phosphate would be expected to bear a

C2-C3-C4 fragment from one source molecule, and a Ct carbon from a second

glycerol molecule (Fig. 2.1).

Significant deviations between the experimental data and the thus calculated values

for a "pure" shikimate pathway (Table 2.3, Box 2.4) cannot be explained within the

framework of commonly known pathways. In particular, biosynthesis of the aromatic

ring from 2-keto-3-deoxyarabino-heptulosonate-7-phosphate (DAHP), which itself

arises from erythrose-4-phosphate and phosphoenolpyruvate via DAHP synthase

(EC 4.1.2.15), predicts that 507c of the c-carhons must not be connected to carbon

atoms that arise from the same source molecule, i.e., f^ {Tyr-e} equals to 0.5 and

yr(2) {Tyr-e} equals 0, but that all the S-carbons must be connected to a carbon atom

from the same source molecule, t.e.,f^ {Tyr-ô} equals to 0 and/^ {Tyr-ft} equals 1

(Fig. 2.8, pattern A). Hence, the detection of tyrosine molecules with intact C ~-C£

fragments (f{T} {Tyr-e} ~ 0.21, Table 2.3; Fig. 2.8, pattern B), and of molecules in

which one of the 6-carbons does not have neighbouring carbons from the same source

molecule (f^ {Tyr-S} ~ 0.12, Table 2.3; Fig. 2.8, pattern C) excludes the sole

operation of the standard shikimate pathway.

2.3. Tyrosine biosynthesis 45

DAHP Tyrosine

1 H H

,

- -»- C=0 \ f 8// 4 C-—C

i CHo4l

'

' HÜ-CH -* HO-C ) C-- r\

! HC-OH

\ HC-OH

C-—c/rH

Ô \H

\ 7lo

^- - - CH2OP032~

Hs-n

\ F ô/

0—c

HO-C^x\y

C-- B\v-//C-—c/f 8 \

H H

H„H

\ t 0 /

i>—C

HO-C ) C-- C\vJ/c-—C/1 8 \

H H

Figure 2.8: Patterns of intact carbon fragments in the aromatic ring of tyrosine.

Thick lines indicate carbon-carbon connectivities arising from a single molecule of glycerol.

Three different pattems of intact carbon fragments (A, B. C) in the aromatic ring are consistent

with the /'values observed at Tyr-ô and Tyr-e. Other patterns would yield /'^ {Tyr-e} ^ 0.50

and/or /f3) {Tyr-ô} + 0.00 and are not compatible with the experimental data (Table 2.3).

Pattern (B) contributes to the observed f{2) {Tyr-e}, pattern (C) to the observed /'*- -1 {Tyr-S}

(Table 2.3). A decomposition of the observed/ values into contributions from (A). (B) and (C)

yields the relative abundance of tyrosine molecules bearing the given pattern of intact carbon

fragments (pattern A = 34 7c pattern B = 43 7c pattern C = 23 7c). Only pattern (A) is in

agreement with the standard shikimate pathway, in which the precursor 2-keto-3-deoxyarabino-

heptulosonate-7-phosphate (DAHP) is synthesized from erythrose-4-phosphate and

phosphoenolpyruvate (compare with Fig. 2.1). The presence of tyrosine molecules with

pattern (B) and pattern (C) thus excludes the possibility that the standard shikimate pathway is

the only pathway leading to tyrosine biosynthesis in //. hispanica (see the text. Table 2.3). The

double line indicates the cyclisation site of the tyrosine ring according to the standard pathway, in

which C, of DAHP is lost as CO-,.

46 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

Table 2.3: Analysis of aromatic 13C fine structures of tyrosine

Relative abund.ances of

ilues carbon atom

indicated intact carbon fragments" at:

Nature of vr

analysed Mid-iexponential phase_

Early stationäry phase

f(X) fW f(i) /(1) /(2) f(3)

Calculated values Tyr-S 0.00 1.00 0.00 0.01 0.99 0 00

Observed values Tvr-6 0.11 0.88 0.01 0.12 0.87 0.01

Calculated values'" Tyr-e 0.50 0.00 0.50 0.50 0.00 0.50

Observed values Tyr-e 0.30 0.22rf 0 48 0.31 0.2ld 0.48

"For the definition of fm, fi2), and f(3) see Box 2.1 and Fig. 1.3

hCalculated with equation 2.4.1 (Box 2.4).

' Calculated with equation 2.4.2 (Box 2.4).''

Derives from tyrosine molecules carrying Ce C*1 fragments originating from a single molecule of glycerol (Fig. 2 8).e

Derives from tyrosine molecules in which one of the 5-carbons does not have neighbommg carbons from the same source

molecule (Fig. 2.8).

Box 2.4: Calculation of the relative abundances of intact carbon fragments in

tyrosine for the shikimate pathway.

In the biosynthesis of the aromatic ring of tyrosine through the common shikimate pathway

(Bentley, 1990; Umbarger, 1978; Voet and Voet, 1995) the ring is assembled from

phosphoenolpyruvate and erythrose-4-phosphate. Phosphoenolpyruvate is assessed via Phe-a and

Tyr-a. Erythrose-4-phosphate is assumed to include an intact Ci-C^-C^ fragment derived from a

first glycerol molecule via the C3-pool of glycolysis, and a C( atom that originates from a second

glycerol molecule (Fig. 2.1) (Rawal et al., 1988). Due to the symmetry of the aromatic ring of

tyrosine the Tyr-Ôx and Tyr-ex carbons give rise to only one C line structure. Therefore,

/('T Tyr-ô) and/^{Tyr-e) are calculated as an average of the/values predicted for Tyr-S i and

Tyr-ô2, or Tyr-e !and Tyr-e2, respectively (Szyperski, 1995):

/^{Tyr-Ô} = 0.5 (Ifm +/(2'n]{ Phe-a} + l/'(1) +/<2<)1 {Tyr-a})

/(2){Tyr-ô} = 0.5 + 0.25 (l/'(1) +f(2*y\[Phe-a} + }/(n +/(>)]{Tyr-a})

/(3){ Tyr-S }=0.00

:(!)/'u'{Tyr-e} =0.50, p->{ Tyr-e }=().()() /'(3){ Tyr-e} =0.50

(2.4.1)

(2.4.2)

2.3. Tyrosine biosynthesis 47

Note that the operation of the oxidative part of the pentose phosphate cycle has not yet been

observed for halophilic archaebacteria. In cell extracts of Haloferax mediterranei and Haloarcula

vallismortis, 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44) was not active at physiological salt

concentrations whereas the observed transketolase and transaldolase activities ensured the

formation of pentoses from hexoses (Rawal et al., 1988). However, even if pentose-biosynthesis in

H. hispanica occurred via glucose oxidation, the same relative abundances of intact carbon

fragments would be predicted for the erythrose-4-phosphate pool.

48 Amino acid biosynthesis m the halophilic archaeon Haloarcula hispanica

2.4. Enzymatic cleavage of surplus threonine

In the additional labeling experiment with [I3C4]-threonine, that was performed to

verify that the threonine pathway for isoleucine synthesis is operational in H. hispanica,

the fine structure of Leu-a (representing C2 of acetyl-CoA, see Fig. 2 in Szyperski,

1995) contains a higher proportion of the doublet component than the fine structure of

Ala-ß (representing C3 of pyruvate), and a large proportion of C-C fragments is

also found for Gly-a (Fig. 2.9). This provides evidence that the exogenously supplied

threonine is cleaved into glycine and acetaldehyde, which would be compatible with the

assumptions that there is threonine aldolase activity in H. hispanica and subsequent

conversion of acetaldehyde into acetyl-CoA (Voet and Voet, 1995). This indication of

threonine aldolase activity in a halophilic archaeon complements data obtained with

other organisms: L-threonine aldolases (E.C. 4.1.2.5) from Saccharomyces ccrevisiae

(GLY1 [Liu ct al., 1997; Monschau et al., 1997]), Escherichia coli (lîaE [Liu et al.,

1998a]) and Pseudomonas sp. strain NCIMB 10558 (ItaP [Liu et al., 1998b]) have been

cloned and expressed, and L-threonine aldolase activity has been demonstrated for

serine hydroxymethyltransferase (EC 2.1.2.1) in E. coli (Schirch et al., 1985), although

this enzyme serves primarily for the cleavage of serine to glycine and a Ct unit. The

serine hydroxymethyltransferase from the thermophilic archaeon Sulfolobus

solfataricus has been shown to possess a/fo-L-tlrreonine aldolase activity (Delle Fratte

et al., 1997). Genome sequencing showed that serine hydroxymethyltransferase is also

present in Methanohaclenum thermoautotrophicum (Smith et al., 1997), in

Methanococcus jannaschii (Bult et al, 1996; Schrumpf et ai., 1991) and in the

hyperthcrmophilic, sulphate-reducing archaeon Archaeoglobus fulgidus (Klenk et al.,

1997).

2.4. Enzymatic cleavage of surplus threonine 49

(A) Lcu-a

to(nC) 545 54 0Ippml

(B) Ala-ß

52 0 51 6

(C) Gly-a

43 2 42 i

Figure 2.9: Evidence for threonine cleavage in H. hispanica derived from the

labeling experiment with f13C4]-threonine.

The ^C-1 'C scalar coupling fine structure of Leu-a (A), which represents C2 of acetyl-CoA,

exhibits a significantly more intense doublet component than that of Ala-ß (B), which represents

C3 of pyruvate. A strong doublet is also detected for Gly-a (C). These data indicate that a fraction

of the cxogenously supplied threonine was clea\ed into glycine and acetaldehyde, with

subsequent conversion of acetaldehyde into acetyl-CoA (Voet and Voet, 1995). The carbon

chemical shifts are relative to those of DSS.

50 Amino acid biosynthesis m the halophilic archaeon Haloarcula hispanica

2.5. Discussion

The exploration of the amino acid metabolism in H. hispanica with

biosynthetically-directed fractional C-labeling revealed that most of the

proteinogenic amino acids are synthesized according to commonly known biosynthetic

pathways. In contrast, the biosynthesis of isoleucine and lysine was found to proceed

through pathways which have not previously been reported for the domain of the

archaea, and the analysis of the labeling pattern of the aromatic ring of tyrosine

indicated yet unknown biosynthetic pathways.

Isoleucine and lysine biosynthesis. In perspective with current knowledge on

amino acid biosynthesis in bacteria and eukarya, the present data on isoleucine and

lysine biosynthesis are particularly intriguing and expand knowledge accumulated for

other organisms to the domain of the archaea. Thus, the identification of a split

threonine/pyruvate pathway for isoleucine biosynthesis in H. hispanica is a novel

finding, since the threonine pathway has not previously been reported for archaea. In

archaebacterial species, such as methanobacteria (Eikmanns et al., 1983: Ekiel et al.,

1983; 1984; 1985a; 1985b; Sprott et al., 1993), and the thermophilic archaeon

Thermoproteus neutrophils (Schäfer et al.. 1989), the pyruvate pathway is used. In

methanogens, isoleucine synthesis can also proceed from propionate or 2-

methylbutyrate (Ekiel et al., 1984). Notably, the threonine pathway is common in

eukaryotic microorganisms and bacteria, although the pyruvate pathway has also been

reported for such species (Dunstan et al.. 1984; Kisumi et al., 1977; Vollbrecht, 1974;

Westfall et al., 1983). However, constitutive, simultaneous operation of the threonine

and pyruvate pathways has so far been observed only in a very few organisms, e.g.,

spirochetes of the genus Leptospira (Westfall et al., 1983). In addition, split isoleucine

synthesis pathways have been reported for eukaryotic and prokaryotic microorganisms

such as Serratia marcescens (threonine/pyruvate pathways) (Kisumi et al., 1977),

E. coli Crookes and K-12 (threonine/glutamate pathways) (LeMaster and Cronan,

1982; Phillips et al., 1972), Rhodopseudomonas sphaeroides (threonine/glutamate

pathways) (Datta, 1978) and Saccharomvces cerevisiae (threonine/pyruvate pathways

or synthesis from homoserine) (Vollbrecht, 1974), but with these organisms a genomic

2.5. Discussion 51

mutation or special growth conditions are required to activate routes other than the

threonine pathway.

Lysine biosynthesis via the dehydrogenase variant of the diaminopimelate

pathway has previously been identified, for example, in Bacillus sphaericus (White,

1983) and in Corynebacterium glutamicum (Ishino et al., 1984; Schrumpfet al, 1991;

Wehrmann et al., 1998), and here we now present direct evidence that this pathway

also exists within the domain of the archaea. It has previously been shown that lysine

biosynthesis in the methanogens Methanospirillum hungatei (Ekiel et al., 1983),

Methanococcus voltae (Ekiel et al., 1985a), Methanothrix concilii (Ekiel et al., 1985b)

and Methanobacterium thermoautotrophicum (Bakhiet et al., 1984) occurs via the

diaminopimelate pathway, but the experiments performed in these earlier studies did

not allow to distinguish between the two variant pathways of Fig. 2.6B. For

Methanobacterium thermoautotropicum the en/ymes that were assayed, i.e.,

dihydrodipicolinate synthase (EC 4.2.1.52) and diaminopimelate decarboxylase (EC

4.1.1.20), catalyze reactions that are common to both variants of the pathway. For

Methanospirillum hungatei (Ekiel et al., 1983), Methanococcus voltae (Ekiel et al.,

1985a), Methanococcus jannaschii (Sprott et al., 1993) and Methanothrix concilii

(Ekiel et al, 1985b), C-labeling experiments with LI- C]acetate, [2- C]acetate or

l3C()2 were used in biosynthetic studies, but with these specifically labeled precursorsI o

both variants of the diaminopimelate pathway (Fig. 2.6B) yield the same positional C

enrichments in lysine (Danson. 1993). Indications for the operation of the

acetylase/succinylase variant in methanogens and Archaeoglobus fuigidus were

obtained from genome sequencing. The gene for L.L-diaminopimelate epimerase (EC

5.1.1.7), which catalyses the conversion of L.L-diaminopimelate to meso-

diaminopimelate in the acetylase/succinylase variant (Fig. 2.6B), was identified in

Methanobacterium thermoautotropicum (Smith et al., 1997), Methanococcus

jannaschii (Bull et al., 1996; Selkov et al.. 1997) ma Archaeoglobus fuigidus (Klenk

et al., 1997), and the succinyl-diaminopimelate desuccinylase gene (E.C. 3.5.1.18) was

found in Methanococcus jannaschii (Bult et al., 1996: Selkov et al.. 1997) and

Archaeoglobus fulgidus (Klenk et al., 1997). The a-aminoadipatc pathway for lysine

synthesis (Fig. 2.6A), which has previously been identified solely for lower eukarya

such as fungi, algae and yeast (Bhattacharjee, 1985; Vogel, 1960). operates in the

52 Amino acid biosynthesis m the halophilic archaeon Haloarcula hispanica

thermophilic archaeon Thermoproteus neutrophils (Schäfer et al., 1989). Overall,

with the new data presented here the implication is that all currently known pathways

for lysine biosynthesis in bacteria and eukarya exist also in the domain of archaea.

Tyrosine biosynthesis. The detected patterns of intact carbon fragments in the

aromatic ring of tyrosine exclude the possibility that the standard shikimate pathway,

in which the C7 precursor (DAFIP) for the ring is formed by condensation of

phosphoenolpyruvate and erythrose-4-phosphate, is the only pathway for tyrosine (and

possibly phenylalanine and tryptophan) biosynthesis in H. hispanica (Fig. 2.8).

Because the patterns of intact carbon fragments in the aromatic ring do not correspond

to the patterns present in those eight metabolic intermediates which are assessed via the

other proteinogenic amino acids (see chapter 1), alternative precursors could not be

identified. Flowever, one can speculate that seduheptulose-7-phosphate is a possible

precursor. The three different patterns of intact carbon fragments in the aromatic ring

of tyrosine (Fig. 2.8) correspond to those that are expected to occur in the

seduheptulose-7-phosphate pool (fragment C2 to C6), assuming that sedoheptulose-7-

phosphate derives cither from two pentoses via transketolase or from erythrose-4-

phosphate and fructose-6-phosphate via transaldolase (Rawal et al, 1988).

Seduheptulose-7-phosphate could be transformed to DAFIP or to a similar precursor to

build up the aromatic ring, and in analogy to the standard pathway Cj of

seduheptulose-7-phosphate would be lost on the biosynthetic route to the aromatic ring

(compare Figs. 2.1,2.8). The data are consistent with the formation of the aromatic ring

by cyclisation of a C7 compound, because one of the 8-carbons of tyrosine is always

connected to a y-carbon that does not stem from the same source molecule. Tyrosine

with intact C ~-CE fragments (Fig. 2.8, patlern B) would then arise from seduheptulose-

7-phosphate molecules with intact C<y-C4 fragments, which themselves derive via

transketolase from the pentoses (compare Fig. 2.1). Tyrosine molecules in which one

Ô-carbon does not have neighbouring carbons from the same source molecule (Fig. 2.8,

pattern C) would derive from seduheptulose-7-phosphate molecules exhibiting both

cleaved CVC^ and C3-C4 connectivities, which are generated when seduheptulose-7-

phosphate molecules initially bearing intact C3-C4 fragments are reversibly

interconverted to erythrose-4-phosphate and fructose-6-phosphate via transaldolase. A

specific labeling experiment with [l,2-,3C2]-ribose instead of [13C\]-glycerol could be

2.5. Discussion 53

performed to further investigate tyrosine biosynthesis in H. hispanica. Ct- C2 units

from ribose would then appear as 13cö-L,Cb units in tyrosine if sedoheptulose-7-

phosphate is the precursor for the aromatic ring. Interestingly, evidence against the

operation of the standard biosynthesis pathways for the aromatic amino acids has also

been obtained from methanogens. For data collected from Methanococcus man'paindis

using a different C-labeling approach, the standard shikimate pathway could also not

account for all the observations (Tumbula et al., 1997), and DAHP synthase (EC

4.1.2.15), which catalyses the condensation of phosphoenolpyruvate with crythrose-4-

phosphate to form DAHP, could not be detected in cell extracts of Methanophilus

mahii (Fischer et al, 1993).

Implications for the evolution of biosynthetic pathways. The present study of

amino acid metabolism in a halophilic archaeon complements those performed with

organisms belonging to other philogenetic groups within the archaea, and confirmed

that most proteinogenic amino acids in archaea are synthesized according to the

common pathways of bacteria and eukarya (Umbarger, 1978; Voet and Voet, 1995).

indicating that these pathways were probably largely established before the divergence

of the three domains (Fig. 2.10)(Woese et al., 1990; Woese, 1998). The threonine and

pyruvate pathways for isoleucine biosynthesis have been revealed to exist in organisms

of all three domains, suggesting that these pathways may as well have arisen before the

three primary lines of descent were formed (Fig. 2.10). Alternatively, a variant

isoleucine biosynthesis pathway that had evolved in one domain might have been

introduced later into early members of the other two domains through lateral gene

transfer (Brown and Doolittle, 1997; Woese, 1998). The acetylase-/succinylase

variants of the diaminopimelate pathway for lysine biosynthesis also exist in many

species of the three domains. In contrast, the diaminopimelate dehydrogenase variant

has been observed only in very few species of the bacteria, and now for the first time

also in a archaeon, and the a-aminoadipate pathway for lysine synthesis has only been

found in lower eukarya and a thermophilic archaeon. The latter two pathways may not

have existed in the universal ancestor. For diaminopimelate dehydrogenase lateral gene

transfer may have occurred at some stage between species of bacteria and archaea, and

the a-aminoadipate pathway may have evolved after the bacteria diverged from the

lineage producing archaea and eukarya. The broad occurence of different amino acid

54 Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica

biosynthesis pathways in all three domains appears to be in line with the observation

that comparative DNA sequencing of many metabolic and biosynthetic enzymes often

provides no clear support for any particular grouping of the domains (Brown and

Doolittle, 1997). In contrast, analysis of the sequences and functions of the proteins

involved in either DNA replication, transcription or translation indicates that archaea

and eukarya have considerable shared ancestry, which solidly distinguishes organisms

of these two domains from those belonging to the domain of the bacteria (Fig. 2.10).

Bacteria Archaea Eukarya

SlimeAnimals

Ravobaetena

Thcrmologa

Aquitex Microsporia

Figure 2.10: Universal phylogenetic tree.

Rooted universal phylogenetic tree as determined from comparathe sequencing of 16S or 18S

ribosomal RNA (Woese, 1987). The data support the separation of the three major domains of

living organisms, the bacteria, the archaea and the eukarya. The evolutionary distance between

two groups of organisms is proportional to the cumulative distance between the end of the branch

and the node that joins the two groups. The root of the tree (shaded rectangle) represents the

position of the universal ancestor of all cells (Woese. 1998). Figure adapted from Madigan et al.,

1997.

2.5. Discussion 55

, fr.»:.».; ft^,!

xa^ ii: ^ijüt> f ^

56

3. Effects of deuterium oxide on the central

carbon metabolism of Escherichia coli

Deuterium labeling has been used to improve the resolution and sensitivity of

protein NMR spectra in a variety of applications. More recently, the use of

2H/13C715N-labelcd proteins in combination with triple resonance experiments has

greatly extended the accessible molecular size range (Gardner and Kay, 1998;

LeMaster, 1994; Salzmann et al., 1998; 1999). To achieve deuterium labeling, proteins

are most often overexpressed in bacteria growing on media containing deuterium oxide

(D20) with either protonated or deuterated carbon sources, depending on the desired

pattern of deuterium incorporation (uniform, random or site-specific labeling)

(Gardner and Kay, 1998).

It has long been known that media enriched in D20 are inhibitory to living

organisms (Katz and Crespi, 1970). Although many bacteria and lower eucaryotes

tolerate complete replacement of hydrogen by deuterium, a reduction of the growth

rate and lower biomass yields are generally observed. The effects of deuterium on

biological systems are manyfold and stem both from solvent isotope effects, resulting

from the change in the isotopic composition of the medium, and isotope effects that

arise from the replacement of hydrogen with deuterium in the CFI bonds of organic

compounds. Deuterium isotope effects influence, for example, the rates of enzyme

catalyzed reactions, ionic equilibria, such as the dissociation of hydrogen/deuterium at

carboxyl and amino groups, the strength of hydrogen bonds and the stability of

biopolymers. Thereby allosteric properties of enzymes and genetic control are affected

(Katz and Crespi. 1970). A large number of studies of deuterium isotope effects on

single in vitro enzyme reactions have been performed as well as a few on in vivo

multienzyme systems (Katz and Crespi, 1970; Saur et al, 1968a; 1968b; Schowen.

1977; Schowen and Schowen. 1982). Given this broad influence of deuterium on many

processes in the cell, the regulatory response of the network of central carbon

metabolism upon addition of D20 to the growth medium is at best difficult to predict.

57

In the present study, the method of fractional C-labeling combined with 2D

[13C, ^-correlation NMR spectroscopy was employed to gain insight into cellular

metabolism during growth in D20-containing minimal media (Fiaux et al., 1999; Sauer

et al., 1997; Senn et al., 1989; Szyperski 1995; 1998; Szyperski et al., 1992; 1996;

1999). In vivo flux ratios were determined for Escherichia coll BL2f(DE3) cells

(Studier and Moffat, 1986) during exponential growth in M9 minimal media with

different concentrations of D20 and protonated glucose as the only carbon source.

E.coli strain BL21(DE3) was chosen because it is frequently used to produce proteins

in deuterated media (e.g.. Venters et al., 1995; Gardner and Kay, 1998). In all

cultivations, 30 % of the supplemented glucose was uniformly 13C-labeled.

A first series of labeling experiments was performed with E. coli cells that had not

been adapted for growth in D20-containing media. Cells taken from a stationary

culture in non-deutcrated M9 medium were used to inoculate M9 media containing

0 %, 30 %, 50 % or 70 ck D20. Subsequently, we performed a second labeling

experiment in 70 °7c D20 with cells that had been adapted for growth in D20 by serial

cultures grown to stationary phase in M9 medium with 0 c7c, 30 % and 50 % D20 (Box

3.f). The following observations were readily apparent. First, the generation time

during exponential growth increases with increasing D20 content (Fig. 3.4). The times

were 1.05 ± 0.04 h for non-deuterated medium, 1.14 ± 0.08 h for medium with 30 %

D20, 1.26 ± 0.08 h for medium with 50 % D20 and 1.50 ± 0.08 h for medium with 70 %

D20. Second, the final cell density in the stationary phase is generally reduced at

higher D20 content (data not shown). Adaptation yielded higher final cell densities,

but did not lead to enhanced growth rates (data not shown). No significant amounts of

secreted metabolites could be detected at the end of the exponential growth phase for

any of the cultivations, indicating that the glucose uptake is balanced with the

production of biomass and the generation of carbon dioxide at all D20 concentrations.

Fractionally C-labeled cells were harvested at the end of the exponential growth

phase, and the dried biomass was hydrolysed in 6M hydrochloric acid. No H/D

exchange occurred during hydrolysis, except at Ô of aspartate. CY of glutamate, and C/

of tyrosine. This could be inferred from a 2D [nC,1H]-FISQC spectrum of fractionally

C-labeled biomass that had been prepared in fully protonated medium and

subsequently hydrolysed in 6M deuterium chloride.

58 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

Box 3.1: Cell growth and sample preparation

Escherichia coli BL21(DE3) cells (Studier and Moffat. 1986), harbouring the pBR322 plasmid for

ampicillin resistance, were cultivated in a minimal medium containing various concentrations of

D20, M9 salts (Sambrook et al., 1989). 2 mM MgS04. 1 uM FeCl3, 10 ml / 1 vitamin mixture

(containing 10 mg / 100ml each of biotin, choline chloride, folic acid, niacinamide, D-pantothenate

and pyridoxal, and 1 mg / 100 ml riboflavin), 5 mg / 1 thiamine, 100 uM CaCl2, 50 uM ZnS04,

4g/l glucose and 50 Lig / ml ampicillin. For fractional C-labeled preparations, 30 % of the

supplemented glucose was uniformly 13C-labeled. For preparations with E. coli cells that had not

been adapted for growth in D20-containing media, M9 medium containing 0 To, 30 %, 50 % and

70 % D20 was inoculated 1:100 with cells from a stationaiy overnight culture in non-deuterated M9

medium. For preparations with adapted cells, M9 medium with 70 % D20 was inoculated with cells

from serial cultures grown to stationary phase in M9 medium with 0 7c 30 %. and 50 % D20. Cells

were grown in 100 ml cultures in 1 1 erlenmeyer flasks at 37 T on a rotary shaker set to 220 rpm.

The total culture volume was 2x 100 ml for non-deuterated medium, 3x 100 ml for medium with

30 % D20, 5x 100 ml for medium with 50 % D20 and 6x ICH) ml for medium with 70 % D20.

Growth was monitored by measuring the optical density at 600 nm (OD600). The cells were

harvested at the end of the exponential growth phase. Secreted metabolites in the growth medium

were assayed with gas chromatography (5890E, Hewlett Packard) on a MD-10 column (Macherey

and Nagel), with butyrate as an internal standard. The dried biomass was hydrolysed in 6M

hydrochloric acid at 110 °C for 24 h. The NMR samples were prepared from dried hydrolysate

and 20 mM DC1 in D20, as follows: solvent volume 600 ul; 90, 140, 170, and 170 mg,

respectively, of the hydrolysate were dissolved for the cultures grown in H20, 30% D2O/70%

H20, 50% D2O/50% H20 and 70% D2O/30% H20.

The relative abundance of intact carbon fragments in the amino acids originating

from a single glucose molecule (/'values, see Fig. 1.3 and Box 2.1) were calculated

from the analysis of the relative multiplet intensities in the l3C-13C scalar coupling

fine structures as described (Szyperski, 1995). using the program FCAL (Glaser,

1999). For non-deuterated samples, the 13C-13C scalar coupling fine structures of the

amino acids were analysed in a 2D [^C^FFj-HSQC spectrum (see chapter 2). For the

samples derived from D20-containing cultures, the presence of multiple deuterium

isotopomers of the amino acids complicated the analysis of the spectra, because the

13C-13C scalar coupling fine structures of different deuterium isotopomers are only

partially separated by the deuterium isotope effect on the chemical shifts (Hansen, 1988)

59

(Fig. 3.2). To resolve the aliphatic C fine structures belonging to sets of deuterium

isotopomers, the following NMR experiments were performed: (i) a 2D ^I-TOCSY-1 ^ I

relayed [ C, H]-HSQC spectrum (Otting and Wüthrich, 1988), (ii) a refocused

2D ^-TOCSY-relayed [13C,1H]-HSQC spectrum, in which the refocusing delay of

the INEPT sequence was tuned to selectively monitor CH groups (Burum and Ernst,

1980), and (iii) a 2D [^C^Hl-HSQC spectrum with 2.5-fold J-scaling (Willker et al.,

1997; Flosur 1990) (Fig. 3.1). For the aromatic carbons a single l3C fine structure was

detected, which could thus be evaluated using a 2D [^C^FFj-FISQC spectrum only.

Spectra recorded for samples containing partially deuterated amino acids were

1 "*

acquired with deuterium decoupling during ?j ( C) using WALTZ-16 (Shaka et al.,

1983). The l3C fine structures were assigned to different sets of deuterium isotopomers

based on previously reported deuterium isotope effects on C chemical shifts (Hansen,

1988), as well as the presence or absence of relay peaks in (refocused) 2D ^-TOCSY-

relayed [13C,JH]-FISQC spectra.

60 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

(A)

lTT

13C

'H

PFG n : (\G, G,

I HI I II MLE\ 17

WALTZ 16

£U.G, G4 G.

Ü

G\RP

G,-

(B)

'H

13/

I I HI I DIPSI '. I I I I MLEV 17

V -y \ -j [ <N

T 1 T 1 I t, I I T I T

GARP

'H

PFG

V\U,IZ16

» 'fl a : a 'ft â 'ÛGl «1 «2 <M <b G G? G6 G6 G7 Gs G8 O,

(C)

lH

13/

61

Figure 3.1: Pulse sequences of the NMR experiments to resolve the C- C scalar

coupling fine structures of partially deuterated amino acids.

(A) 2D ^I-TOCSY-relayed [nC,JHl-HSQC experiment (Oiling and Wüthrich, 1988). extended

with a z-filter to obtain pure in-phase C- C scalar coupling tine structures (Kover et al,

1993).

(B) Pulse sequence of the refocused 2D 'H-TOCSY-relayed l^C^Hl-HSQC experiment. The

period 2t in the refocused INEPT sequence (Burum and Ernst. 1980) is adjusted to l/(2Jriî) for

selective observation of CH groups. Proton decoupling during t\ is achieved using the composite

pulse decoupling scheme D1PSI-2 (Cavanagh and Ranee. 1992; Shaka et al., 1988). To obtain

pure in-phase C- 'C scalar coupling line structures, /-filters are placed on each side of the t{

evolution period and before acquisition. For (A) and (B) isotropic mixing is performed with the

MLEV-17 sequence (Ba\ and Davis, 1985). The phase c\c1e is <)>, = {4y. 4(-y)}, <|)2 = {2x. 2(-x)}.

<|>3 = {x, -x}. and (j)rec = {\, -x, -x. x. -x, x,x. -x}. Pulsed field gradients were employed for

coherence pathway rejection (Bax and Pochapsky, 1992; Wider and Wüthrich, 1993), and a 2 ms

spin-lock pulse (Otting and Wüthrich, 1988) was used to purge the magnetization arising from

C-bound protons and from the residual "HOH signal. C-decoupling during t2 and ~H-

decoupling during t\ were achieved using GARP (Shaka et al.. 1985) and WALTZ-16 (Shaka et

al., 1983), respectively. Quadrature detection in CO] was accomplished with States-TPPI (Marion

et al., 1989).

(C) The t{ time element for J-scaling (Willker et al.. 1997; Hosur, 1990). The ^C-1 'C coupling

evolves during the full t\ period (Jfjc)- whereas the C chemical shift evolves only during the

period denoted Hq. This version scales up the LlC-LV coupling by a factor of 2.5. The J-scaling

evolution time element was introduced into the pulse sequence for 2D L^C^Hl-HSQC spectra

(Bodenhausen and Ruben. 1980).

62 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

(A) .

A

; Asp-ß•

k

A

•

A

•

nA

9

A i•

'ill

• -CUH- CpH2-A-CaD-CPH2-

A

A

*

Ji JiMl.*

i'

i

co(13C) 36.8 36.2 [ppm]

(C) A Asp-ß• -CnH-C(iH2-a -C"D-CPH2-O -C^H-C^HD-

d -C"D-CPHD-

d a a m do d a

o oooo ooo o

36.5 35.9

(B) • Asp-ß

0

•

•I* »

J

J u ^illlL«I | I | I

36.8 36.2

(D)O

o

o

o

o

o Asp-ß

o

oo

36.5 36.0

(E) Thr-y

-CpH-C'HD2-CPD-CYHD2

21.7 21.2

63

Figure 3.2: Illustration of the use of the different C- H correlation experiments

for observation of the C- C scalar coupling fine structures of different

aspartate and threonine deuterium isotopomers obtained from E. coli cells

grown on a M9 medium in 70 % 1)20.

All spectra were recorded at a C resonance frequency of 125.8 MHz with a Bruker DRX500

spectrometer, except that (C) was recorded at a l~C resonance frequency of 188.6 MHz with a

Bruker DRX750 spectrometer, fn all the panels the components of the multiplet structures

originating from the different deuterium isotopomers are identified by symbols that are explained

by the inserts in the panels (A), (C) and (E).

(A) Overlapping Asp-ß line structures of the C^tF-C^H ant' l'1c C^TF-C"!) isotopomers in a

2D ["C'HJ-HSQC spectrum.

(B) Fine structure of the CPH2-C°H isotopomer. observed on Hu in the 2D ^-TOCSY-relayed

[13C,'H1-HSQC spectrum.

(C) Asp-ß fine structures obtained from a 2D [lC,'H)-HSQC spectrum. The C%2 isotopomers

dominate and only trace amounts of the C^TTD isotopomers are visible.

(D) Resolved fine structure of the QHD-CaH isotopomer, observed on Ha in the refocused

2D ^I-TOCSY-relayed [ 'WflFHSQC spectrum, selective for CH groups. Note the differences

in the relative multiplet intensities compared to the C H2 -C('H isotopomer. The star denotes an

impurity.

(E) Resolved fine structures of the C^EDy-C^D and CYHD2-C'5H isotopomers in the

2D 113C,'H]-HSQC spectrum with J-scaling by a factor of 2.5, The deuterium isotope effect on

Ca causes additional splitting of the multiplet components. Note the more intense doublet

component in the OD isotopomer, which indicates a higher fraction of intact CY-C> fragments

from the same glucose molecule.

The acquisition parameters for the 2D ['"VJni-HSQC spectra were as described in Chapter 2

(Box 2.1). For the (refocused) 2D 'H-TOCSY-relayed [nC.'H]-HSQC spectra, the 13C-canier

was set to 42.0 ppm relative to DSS. The sweep width v.as 67.5 ppm. and the measurement time

was 40 h per spectrum (3"000 x 4'096 complex points; f)rrm = 353 ms; /2max = 682 ms. 8 scans

per increment, relaxation delay between two scans 2 s). For the J-scaled 2D [^C^HFHSQC

spectrum the 13C-carrier was set to 37.0 ppm relative to DSS and the sweep width was 16.9 ppm.

The measurement time was 11 h per spectrum (F5Ü0 x 4'096 complex points; rlmax = 353 ms;

^2max = 6l4 ms- 4 scans Per increment, relaxation delay between two scans 2 s). Before Fourier

transformation the time domain data were multiplied in t{ and t2 with squared sine windows

shifted by Tt/2. The digital resolution after zero-filling was TO Hz/point along C0j and

0.7 Hz/point along m2 for the (refocused) 2D lH-TOCSY-relaycd I^C'HJ-HSQC spectra, and

1.4 Hz/point along co, and 0.8 Hz/point along co2 for the J-scaled 2D [13C.1H1-HSQC spectrum.

64 Effects of deuterium oxide on the central carbon metabolism of Escherichia coli

The /values of the amino acids represent the patterns of intact carbon fragments in

the metabolic intermediates from which they are synthesized. Analysis of the /values

was performed as described (chapter 2; Szyperski. 1995) and revealed agreement with

standard biosynthesis pathways (Neidhardt et al., 1996; Voet and Voet, 1995). The

13C~13C scaiar coupling fine structures of different deuterium isotopomers of the

amino acids could be resolved (Fig. 3.2; Table 5.2, appendix). It was found that for

some of the proteinogenic amino acids the/ values depend on the dcuteration of a given

carbon atom (Fig. 3.3). This is observed when the metabolic precursor of the respective

amino acid is synthesized via two alternative pathways (see Fig. 3 in Szyperski, 1995),

so that different C isotopomers introduced through the alternative pathways correlate

with different degrees of dcuteration (Fig. 3.3). In case no H/D-cxchange takes place at

the respective carbon atom on the biosynthetic route from the intermediate to the amino

acid, this correlation is preserved and accessible through analysis of the amino acids

(Fig. 3.3). For the group of amino acids synthesized from oxalacetate (aspartate,

threonine and methionine), the/values depend on the deuteration of C^ and thus reveal

distinct patterns of intact carbon fragments, and hence different metabolic origins for

individual C^ deuterium isotopomers (Figs. 3.2, 3.3; Table 5.2). H or D at Ccx and also

at CY of threonine is introduced during synthesis from oxalacetate (Voet and Voet,

1995), and therefore the / values are independent of deuteration at these carbon

positions (Fig. 3.3). Since H/D exchange occurs at O of aspartate during hydrolysis in

hydrochloric acid, the Asp OH2 isotopomer dominates after hydrolysis albeit trace

amounts of Asp ChHOD can be detected (Fig. 3.2). Consequently, the/ values detected

for the Asp C^H2 isotopomer represent virtually the whole oxalacetate pool, whereas

the / values of Asp OHD selectively represent the pool of oxalacetate molecules

deuterated at C}. The non-deuterated oxalacetate pool can not be selectively assessed.

The Thr OH isotopomer represents both non-deuterated and mono-deuterated

isotopomers of oxalacetate. and methionine can not be quantitatively assessed, because

the 13C fine structures of the different O deuterium isotopomers strongly overlap.

Dependence of the /values on deuteration is also observed for the amino acids derived

from the C[-metabolism. For serine, the relative abundance of intact Ca-C^ fragments

depends on dcuteration of C^, while for glycine, the / values depend on the deuteration

of Ca (Table 5.2, appendix). In contrast, for the amino acids synthesized from ribose-

5-phosphate, erythrose-4-phosphate, phosphoenolpyruvate, pyruvate and acetyl-CoA

TCAPEP carboxylase(anaplerotic pathway)

Oxalacetate

Transamination

Aspartate

+H,NC H/D

^ 0= :CaI

Figure 3.3: Correlation of intact carbon fragments in aspartate with the deute¬

rium labeling pattern.

Oxalacetate is synthesized via two alternative pathways, each of which may generate molecules

that have different patterns of intact carbon fragments as well as different degrees of deuteration.

Intact C2-C3 fragments in oxalacetate solely arise from Pep via the anaplerotic pathway;

molecules deuterated at C3 are preferentially generated via the TCA cycle, while the anaplerotic

reaction introduces a larger fraction of non-deuterated species (see the text and Table 3.1). In the

biosynthesis of aspartate from oxalacetate via a transaminase, no H/D exchange occurs at CF\

while H or D are introduced at C". Consequently, the pattern of intact carbon fragments observed

for aspartate correlates with the deuteration of O.but not with the deuteration of C((.

Asp C^HD/D2 exhibits a larger fraction of cleaved Cu-d3 connectivities than Asp L]R2. Note

that H/D exchange at Ö during sample preparation complicates data analysis of aspartate, as

described in the text. Thick C-C bonds: intact carbon fragments originating from a single source

molecule of glucose. Dotted C-C bonds: cleaved C-C connectivities. Thin C~C bonds: not

specified here. Pep: phosphoenolpyruvate.

66 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

the / values are independent of the deuteration pattern and correspond to the values

obtained from preparations in fully protonated medium. Likewise, the / values

observed for the amino acids synthesized from 2-oxogfutarate are independent of

deuteration, and correspond to the pattern of intact carbon fragments in oxalacetate (ah

isotopomers, assessed from Asp OFF) and acetyl-CoA (assessed from Leu-a). This is

due to the fact that 2-oxoglutarate is exclusively formed by irreversible condensation

of oxalacetate with acetyl-CoA in the tricarboxylic acid cycle (TCA).

The evaluation of the patterns of intact carbon fragments in the pools of the eight

principal intermediates that link primary metabolism to amino acid biosynthesis

determines their metabolic origin and yields information on active biochemical

pathways and flux ratios at several key points in central metabolism (Table 3.1, Fig.

3.5, Box 3.2) (Sauer et al.. 1997; Szyperski, 1995: Szyperski et al., 1996, 1999). The

present data show that no additional metabolic pathways are induced or inactived due

to the presence of D20, i.e., the topology of active pathways remains unchanged.

Moreover, cells that had been adapted for growth in D20 exhibit the same response to

the presence of D20 in the nutrient medium as non-adapted cells. With regard to the

three main processes, the flux ratios characterizing glycolysis and the pentose

phosphate phosphate pathway are not measurably affected by the addition of D20

(Table 3.1, Fig. 3.5). In sharp contrast, the anaplerotic supply of the TCA cycle via

carboxylation of phosphoenolpyruvate increases relative to the influx of acetyl-CoA at

higher D20 contents, i.e., a larger fraction of the oxalacetate pool (all isotopomers) is

synthesized via the anaplerotic reaction (Table 3.1, Fig. 3.5), as evidenced by the

increasing fraction of oxalacetate molecules with intact C2-C3 connectivities. The

dependence of the patterns of intact carbon fragments in oxalacetate on deuteration at

C3 reveals different metabolic origins and fluxes for the individual deuterium

isotopomers in the metabolic network. Threonine and aspartate molecules deuterated at

Cß (Thr CßD, Asp CPHD and CßD2) exhibit a lower fraction of intact Ca-Cp

connectivities than Asp OH2, which represents all isotopomers in the total oxalacetate

pool. Oxalacetate deuterated at C; is therefore preferentially generated via the TCA

cycle (Table 3.1), while the anaplerotic reaction introduces a larger fraction of non-

deuterated species, suggesting that the metabolites in the TCA are more highly

deuterated than those in glycolysis. This can also be inferred from the analysis of

67

serine. The Ser CPH2 isotopomer clearly dominates at all degrees of deuteration, and it

derives to a large extent directly from 3-phosphoglyccrate since only a minor fraction

has been reversibly mterconverted to glycine and a Cj unit via serine

hydroxymethyltransferase (Table 3.1, Fig. 3.5). This indicates that C^ of

3-phosphoglycerate is highly protonated, because H or D that appears at serine O is

not introduced or exchanged on the biosynthesis route from 3-phosphoglycerate to

serine. The Ser OHD isotopomer is present only in small amounts, and a higher

fraction of this isotopomer derives from glycine and a Cj unit (Table 3.1). The C|

metabolic pathways generating glycine are also affected by the presence of D20. The

Gly CaHD isotopomer directly derives from the C'~Ca fragment of serine via serine

hydroxymethyltransferase at all D20 levels, whereas a significant fraction of the CaH2

isotopomer arises from a different pathway at high D20 concentrations, e.g., from C02

and a C[ unit through the reversible glycine cleavage system. Alternatively, the

threonine degradation pathway may contribute to the generation of the Gly CCXH2

isotopomer at high D20 contents (Voet and Voet, 1995).

68 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

Table 3.1: Origin of intermediates in the central carbon metabolism of E.coli

during exponential growth in non-deuterated and deuterated media0

average fraction of total pool [%(%,-\b

metabolites non-deuterated

M9 medium

deuterated

M9 mediun/

(70 % D-,0)

Pentose Phosphate Pathway

Pep originating pentoses (upper bound) 23 + 5 15 ±5

Ri5P originating from GAP + C2 (TK reaction)

Ri5P originating from E4P (TK and TA reaction)

E4P originating from fructose (lower bound)

70 ±2

9± 2

44 ± 5

73 + 2

7 ±2

44 ±5

Glycolysis

Pep originating from Oa

Pyr originating from malate (upper bound)

Pyr originating from malate (lower bound)

5 ±3

0-3

0-2

6±4

0-4

0-2

Tricarboxylic acid cycle

Oa originating from Pep

total Oa (Oa deuterated at C3)fl

Oa reversibly intcrconverted to fumarate

1c originating from glyoxylate and succinate

36 ± 1

38 + 10

0-5

51 ±3 (27 ±3)

40 + 7

0-3

Ct metabolism

Ser originating from Gly + C j

SerChl2(SerCßUD)Gly originating from C02 + Cf

Gly Caih (Glv CaHD)

28 ±2

0-3

23 ± 3 (52 ± 2)

13±3£'(0-3)

" The fractions wcie calculated with the equations gnen in Box 3 2 The abbreviations for the metabolites are given in the

legend of Fig 3 5 In addition. TK and TA denote transketolase and transaldolase. respectively.hThe numbers describe the fraction of the total pool of the metabolite indicated in italics that originates from the specifiedpathway, oi has undergone the specific reversible mlerconversion reaction Some of these flux ratios also appear in Fig 3 5.

Except for Oa originating from Pep, the fractions turned out to be identical w ithin the experimental error for all cultivations.'The fractions are avciage values obtained from cultivations ot both non-adapted and adapted cells

''Fractions at 30 % D20: 40 ± 2 (6 ± 2); fractions at 50 7c D20 44 ± 2 ( 17 ± 2).

cNote that the threonine degradation pathway (Voet and Voet, 1905) may also contribute to the observed fraction.

rel

1.5 -

(A) E. coli generation time

1.4 -

1.3 -

~r /

1.2 -

1.1 -

1.0 -

I ' I ' I""

' I ' !

0.0 20.0 40.0 60.0 80.0

% D20 in H20

'rel

1.5 /ß\ Anaplerotic synthesisof oxalacetate

^ r

0.0 20.0 40.0 60.0 80 0

% D20 in H20

Figure 3.4: Increase of the generation time and the anaplerotic supply of the TCA

with increasing D20 concentrations in the growth medium.

(A) Increase of the relative generation time. Tro( of E.coli BL21(DE3)pBR322 cells during

exponential growth in M9 minimal media. The generation times during exponential growth were

1.05 ± 0.04 h for non-deuterated medium. 1.14 ± 0.08 h for medium with 30 % D20, 1.26 ± 0.08

h for medium with 50 % D20 and 1.50 ± 0.08 h for medium with 70 % D20 (adapted and non-

adapted cells).

(B) Increase of the fraction of oxalacetate (total pool) synthesized via carboxylation of

phosphoenolpyruvate (anaplerotic reaction). Crel. with increasing D20 content of the nutrient

medium (Table 3.1). In (A) and (B). the experimental error of the relative values was calculated

from the experimental error of the absolute values using the Gaussian law of error propagation.

70 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

Glycolysis

Glc COo Pentose-Phosphate Pathway

Tricarboxylic acid cycle

71

Figure 3.5: Central carbon metabolism and ratios of fluxes of E.coli BL21(I)E3)

growing in non-deuterated M9 medium and M9 medium with 70 % D20.

The fractions of molecules (in 7c) given in square boxes arc synthesized via the fluxes pointing

at them. Numbers in ellipses indicate the amount of reversible interconversion of the

molecules. Fractions in plain numbers are from preparations in non-deuterated medium, fractions

in bold numbers from cultivations in medium containing 70 % D20. The metabolic flux ratios in

glycolysis and the pentose phosphate pathway do not change significantly upon addition of D20

to the growth medium. In contrast, the anaplerotic supply of the TCA cycle via carboxylation of

phosphoenolpyruvate increases with increasing D20 le\els (fluxes marked in bold). Furthermore.

the presence of D20 affects the C| metabolic pathways generating serine and glycine (see Table

3.1). Note that the indicated fractions do not all have the same precision (Table 3.1).

Abbreviations: Glc. glucose; G6P, glucose-6-phosphaie; F6P, fructose-6-phosphate; Ru5P.

ribulose-5-phosphate; Ri5P. ribose-5-phosphate; Xu5P, xylulose-5-P; S7P, sedoheptulose-7-

phosphate; E4P, erythrose-4-phosphate; G3P, glyceraldehyde-3-phosphate; 3Pg.

3-phosphoglycerate; Pep, phosphoenolpyruvate; Pyr. pyruvate; AcCoA. acetyl-CoA; Oa.

oxaloacetate; 20g. 2-oxoglutarate; Mai. malate; Finn, fumarate; Sue. succinate; Ser. serine;

Gly, glycine.

72 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

The influence of deuterium on metabolism is on a first level determined by its effect

on enzymes, which comes (i) from solvent effects, leading to, e.g., replacement of

hydrogen by deuterium at exchangeable positions, such as the carboxyl, amino and

hydroxy 1 groups of amino acids at active sites, (ii) from incorporation of deuterium at

non-exchangeable positions in the enzymes, and (iii) from the occurence of deuterated

substrates and cofactors. On a second level, deuteration effects allosteric properties of

enzymes and control mechanisms in general (Katz and Crespi, 1970). Most remarkably,

we found that despite of the manyfold effects of deuterium on the enzymes and on cell

physiology in general, the influence on the flux distribution in the central metabolic

network of E. coli BL21(DE3) is only very limited, and is, under the presently chosen

growth conditions, restricted to a change in the regulation of the TCA cycle (Table 3.1,

Fig. 3.5). Furthermore, the C| metabolic pathways generating serine and glycine are

affected.

The observed relative increase in the anaplerotic supply of the TCA is correlated

with the increase in generation time (Fig. 3.4). However, since the total biomass

production rate is reduced at higher D20 levels, one would expect that also the

anaplerotic supply of the TCA decreases. Relatively increased anaplerosis thus suggests

that the presence of D20 represses the TCA cycle and respiration more strongly when

compared with glycolysis and the pentose phosphate pathway. An inhibition of the TCA

cycle would be consistent with the observed increase in generation time, since D20 may

limit its functions for energy generation and the production of precursors such as 2-

oxoglutarate, which is an essential intermediate both for the synthesis of amino acids

and the nitrogen metabolism of the cell in general (Neidhardt et al, 1996; Voet and

Voet, 1995). In vitro studies showed that several enzymes of the TCA are significantly

inhibited both by D20 solvent effects and by deuterated substrates, i.e. succinate

dehydrogenase (Thomson and Klipfel. I960; Laser and Slater. I960), malate

dehydrogenase (Thomson et al., 1962), isocitrate dehydrogenase (Coleman and Chu,

1969), fumarase (Thomson, 1960) and aconitase (Thomson et al, 1966; Thomson and

Nance, 1969). The present study indicates that the metabolites in the TCA are more

highly deuterated than those in glycolysis when E.coli cells are grown in D20-

containing minimal media with protonated glucose as the carbon source. D20 inhibition

may thus possibly limit the flux through the TCA cycle, yielding increased levels of

73

acetyl-CoA if the pentose phosphate pathway and glycolysis are much less affected.

Since phosphoenolpyruvate carboxylase is activated by acetyl-CoA (Canovas and

Kornbcrg, 1965), the excess glycolytic intermediates could then in part be diverted

through the anaplerotic pathway.

It has previously been observed that the supply of the TCA cycle in E. coli cells

markedly depends on the aeration of the fermentor, with decreasing contribution of

anaplerosis at strong aeration most probably due to the increased TCA flux (Wüthrich et

al, 1992; Szyperski, 1995). Given the evidence that cell growth in D20-containing

media is in part affected by the inhibitory effect of D20 on the TCA cycle, strong

aeration should be assured during production of deuterium labeled proteins in D20-

containing media, so that a maximal TCA flux is achieved, which possibly increases the

yield of protein production.

74 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

Box 3.2: Origin of intermediates in the central carbon metabolism: Calculation of

the fraction of molecules synthesized via a given pathway from the relative

abundance of intact carbon fragments in the amino acids.

The equations are derived based on the principles defined by Szyperski (1995). For the definitions of

the/values see Fig. 1.3 and Box 2.1.

|j(0 +/(2^] {Phe-a, Tyr-a}. e.g., represents the average value of [/(1) + f {2>] {Phe-a} and

If (1)+/(2*>1 {Tyr-a}.

Glycolysis

Phosphoenolpyruvate originating from oxalacetate (phosphoenolpyruvate carboxykinase).

Phosphoenolpyruvate is assessed via Phe and Tyr; oxalacetate (total pool) via Asp

(O H2 isotopomer, see the text)

ppp-

/(2>){Phe-a, Tyr-u}r crOa ~

p-n ß

Pyruvate originating from malate (malic enzyme) (upper bound). Pyruvate is assessed via Ala.

Malate is not directly assessable. A boundary value for [/'(-') +./'(~''1] {malate-C2} is represented

by l/'(l) +/'<2x)l {Asp-a(C%2), Asp-ß(CTH2)}), assuming complete equilibration of the malate

and oxalacetate pools. This yields an upper bound for the fraction of pyruvate derived from

malate:

PYR =

l/(l) + /(2',)l{Ala-a}-[/') + /y)l{Phe-a.Tyr-a}Mai.ub (M PM ft ß Ml Pn

If +f l{Asp-u(CH2).Asp-ß(r H>)}-[/( '+ r ']{Phe-a, Tyr-a}

Pyruvate originating from malate (lower bound). The upper boundary value for [f(l^ + f l-2^]

{malate-C2} equals 1, when no exchange between the malate and oxalacetate pools takes place.

This yields a lower bound for the fraction of pyruvate derived from malate:

pyr -

l/"(l) + ./(y)HAld-(x}-[/')+/(")]{Phe-u. Tvr-q}

•-!./ +/ ]{ Phe-a. Tyr-a}

Box 3.2 (continued)

Pentose phosphate pathway

Phosphoenolpynivate originating from pentoses (upper bound). Pentoses are assessed via His.

1 ./l2){Phe-a,Tyr-a}Ppppgp = ,, , :

, , , . ,

' ' '

"

2 1 (i) O) /2){Phe-a. Tyr-u}m, /*(2){His-8}5 5 /u){His-6}

f^ {Phe-a, Tyr-a}ppp represents intact carbon fragments in the Pep molecules synthesized

glycolysis or the pentose phosphate pathway (for the definition of PEP0a, see above):

(2) /(2){Phe-a,Tyr-rt}-PEP0a-/2){Asp-a(CPH,)}./ ;{Phc-a.lyr-a}pPP = —

JZpFiP~ _—_—

Ribose-5-phosphate originating from glyeeraldehyde-3-phosphate and a C2 unit.

Transketolase reaction.

Ri5p0AP.c2 = l/(1) + ./(2)HHis-ß}

Rihose-5-phosphate originating from erythrose-4-phosphate.

Transketolase and transaldolase reactions

Ri5PE41, = /(1){His-8}

Erythrose-4-phosphate originating from fructose-6-phosphate (lower bound).

Erythrose-4-phosphate is assessed via Tyr.

F4P. -

/('){Tyr-r}R5P + /2){Tyr-r}-,/(')(Tyr-i-}-/(2){Tyr-e}R5rJ

F6P ( I'M ( 5 1

with

/(1){Tyr-e}R5p = 0.5(/(1){His-u} +f/(,) + /(2)l{His-ß})

and

/2){Tyr-c}R5P = 0.5([./(2) + /")l{HiS-a} + [/(") + ./f3)){His-p})

76 Effects of deuterium oxide on the central carbon metabolism ofEscherichia coli

Box 3.2 (continued)

Tricarboxylic acid cycle

Oxalacetate originating from phosphoenolpvruvate (total pool) (phosphoenolpyruvate carboxylase;

anaplerotic reaction). The total pool of oxalacetate is assessed from Asp (O H2 isotopomer).

-

I f<2) + f0)HAsp- a(CPH2), Asp- ß(CpH,)>Pep

~

i~>\ (T.)

1/ +/ 'l{Phe~u. Tyi-a}

Oxalacetate originating from phosphoenolpxruvate (deuterated isotopomers). Deuterated

oxalacetate (C3HD and C^D2) is assessed from Asp (C"HD isotopomer; see the text).

rv, -

l/2)+f0)l{Asp-ß(Cr>HD)}waPep

-

p\ pi

[f +fl 'UPhc-u. Tyi-ot}

Oxalacetate reversibly mterconverted to fumarate (total pool).

0aPe/0){Asp-ß(CßH7)}

ep

/0){Asp-ß(CPH2)}+/0){Asp-a(CPH,)}

Isocitrate originating from glyoxylate and succinate (isocitrate lyase). Isocitrate is assessed from

Glu and Pro.

l/(1)+ /(2^]{Glu-u, Pro-a}-[/(1)+ /(2 )l{Asp-a(CßH2), Asp~ß(CpH\)}

l-I/,(1)+Â(2*)HAsp-u(CpH1), Asp-ß(CPH2)}

C! metabolism

Serine originating from glycine and a Cj unit. The patterns of intact carbon fragments in the

glycolytic precursor of Ser (and Gly). 3-phosphoglycerate. are equal to those observed for

phosphoenolpyruvate.

cw -

l/-(') + /'2')]{Phc-a.T)r-a}-/(1){Sei-ß}ociG,

-— ——

If +/l(Phc-a, Tyr-a}-1

Glycine originating from C02 and a Cj unit.

filv -

[/(1)+/(2)]{Phe-a.T>i-a}-/(n(Cdy-a}vjiyC02

- —— —

ir"+f-,]{Phc-a. Fyi-a}-1

77

/*~ V

5 s

is .1 r

78

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f^. tfflSSTK^ JA4- y s'¬

il w*J"'„ .

89

5. Appendix

5.1. Amino acid biosynthesis in Haloarcula hispanica

Table 5.1: Relative intensities of C multiplet components and derived relative

abundance of intact C2 and C3 fragments in the amino acids.

90 Appendix

Table 5.1: Relative intensities of 13C multiplet components and derived relative

abundance of intact C2 and C3 fragments in the amino acids.

relative abundance of intact

carbon fragments'7relative intensities of multiplet

Observed

carbon position

preparation^ components"

h 'd

Terminal carbons:

Ala-ß me 0.12 0.88

es 0.11 0.89

Arg-ô me 0.11 0.89

es 0.11 0.89

Gly-a me 0.09 0.91

es 0.09 0.91

His-82 me 0.08 0.92

es 0.09 0.91

Ile-Y2 me 0.13 0.87

es 0.11 0.89

Ile-5 me 0.32 0.68

es 0.33 0.67

Leu~Sj me 0.15 0.85

es 0.11 0.89

Leu-S2 me 0.87 0.13

es 0.87 0.13

Lys-e me O.ll 0.89

es 0.11 0.89

Pro-0 me 0.11 0.89

es 0.11 0.89

Ser ß me 0.51 0.49

es 0.54 0.46

Thr-Y2 me 0.48 0.52

cs 0.50 0.50

Val-Yi me 0.11 0.89

es 0.11 0.89

Val-Y2 me 0.83 0.17

es 0.87 0.13

r(l) AT)

0.05 0.95

0.05 0.95

0.04 0.96

0.05 0.95

0.02 0.98

0.02 0.98

0.00 1.00

0.03 0.97

0.07 0.93

0.04 0.96

0.31 0.69

0.32 0.68

0.09 0.91

0.04 0.96

1.00 0.00

0.99 0.01

0.04 0.96

0.05 0.95

0.04 0.96

0.04 0.96

0.55 0.45

0.58 0.42

0.51 0.49

0.53 0.47

0.05 0.95

0.04 0.96

0.95 0.05

0.99 0.01

"

/, singlet; /d doublet split by the smaller coupling; /j. doublet split by the larger coupling; Jdd doublet of doublets; /, triplet.(Fig. 1.3)

Experimental error: ±0.02

'me: sample harvested in the mid-e\ponential phase, cs: sample harvested in the early stationary phase.Not evaluated due to an insufficient sienal-to-noise ratio.

91

Table 5.1: Relative intensities of C multiplet components and derived relative

abundance of intact C2 and C3 fragments in the amino acids.

Observed preparation''carbon position

relative intensities of multipletcomponents0

/. T h- dd

iclative abundance of intact

carbon fragmentsb

r(l) c(2) r(2 ) :(3)p..

Central, carbons in C3 fragments

(different scalar coupling):

Ala-a me

es

Asp-a me

es

Asp-ß me

es

Glu-a me

es

G1u-y me

es

His-a me

es

His-ß me

es

Ile-a me

es

Leu-a me

es

Mct-a me'

es

Phe-a me

es

Phe-ß me

es

Pro-a me

es

Ser-a me

es

Thr-a me

es

Tyr-a me

es

Tyr-ß me

es

Val-a me

es

0.07 0.01 0.03 0.88 0.00 0.01 0 04 0.95

0.08 0.01 0 03 0.88 0 00 0.01 0 05 0.94

0.18 0 09 0.32 0.41 0.16 0.09 0.37 0.38

0.19 0 09 0.33 0.39 0.18 0.08 0.38 0.36

0.18 0.34 0.31 0 17 0.16 0.39 0.36 0.09

0.19 0.32 0.32 0 17 0.18 0.37 0.37 0.08

0.18 0.34 0.31 0.17 0.16 0.40 0 36 0.08

0.19 0.33 0.32 0 16 0.18 0.39 0.38 0.07

0.09 0.02 0.77 0.12 0.04 0.00 0 96 0.00

0.09 0.02 0.77 0.12 0.03 0.01 0.95 0.01

0.08 001 0.00 0 91 0.01 0.01 0 00 0.98

0.08 0.00 0.00 0 92 0.00 0.00 0.00 1.00

0.07 0.80 0.01 0.12 0.0L 0.99 0 00 0.00

0.08 0.79 0.01 0.12 0.02 0.98 0.00 0.00

0.18 0.03 0.70 0.10 0.16 0.00 0.84 0.00

0,18 0.03 0.69 0.10 0.17 0.00 0.83 0.00

0.09 0.02 0.77 0.12 0.04 0.01 0.95 0.00

0.09 0.03 0.77 0.11 0.03 0.02 0.95 0.00

0.17 0.09 0.31 0.43 0.14 0.09 0.36 0.41

0.08 0.04 0.00 0.88 0 00 0.05 0.00 0.95

0.08 0.01 0.00 0.91 001 0.01 0.00 0.98

0 07 0.81 0.01 0.12 0.00 1.00 0.00 0.00

0 07 0.79 0.02 0.12 0 01 0.98 0.01 0.00

0 17 0.36 0.33 0.14 0.15 0.42 0.38 0.05

0.17 0.35 0.31 0.17 0.15 0.41 0.36 0.08

0 08 0.00 0.43 0.49 0.01 0.00 0.53 0.46

0 08 0.01 0.45 0.46 0.01 0.00 0.56 0.43

0.17 0.10 0.32 0.41 0.15 0.10 0.37 0.38

0.18 0.10 0.32 0.40 0.16 0.10 0.37 0.37

0 07 0.01 0.00 0.92 0.00 0.01 0.00 0.99

0 08 0.00 0.00 0.92 0.01 0.00 0.00 0.99

0 07 0 81 0.01 0.12 0.00 1.00 0 00 0.00

0 07 0.80 0.01 0.12 0 01 0.99 0 00 0.00

0.08 0.01 0.79 0.12 0.02 0.00 0.98 0.00

0 08 0.03 0.77 0.12 0.02 0.03 0 95 0.00

92 Appendix

Table 5.1: Relative intensities of 13C multiplet components and derived relative

abundance of intact C2 and C3 fragments in the amino acids

relative: intensities of multiplet relative: abund;:ince of intact

Observed preparation' components" carbon fragments

carbon positionc hi h f(0 f(2) fO)

Central carbons in 67} fragments

(equal scalar couplings):

Arg-ß me 0.41 0.52 0.07 0.50 0.50 0.00

es 0.42 0.51 0 07 0.51 0.49 0.00

Glu-ß me 0.42 0.51 0 07 0.51 0.49 0.00

es 0.44 0.50 0.06 0.54 0.46 0.00

lle-Yi me 0.30 0 60 0.10 0.33 0.65 0.02

es 0.32 0.60 0 08 0.36 0.64 0.00

Leu-ß me 0.75 0.22 0 03 0.99 0.00 0.01

es 0.76 0.22 0.02 1.00 0.00 0.00

Lys-ß me 0.17 0 67 0.16 0.15 0.78 0.07

es 0.19 0 67 0.14 0.18 0.77 0.05

Lys-Y me 0.44 0 50 0.06 0.54 0.46 0.00

es 0.43 0.51 0.06 0.53 0.47 0.00

Lys-8 me 0 08 081 0.12 0.02 0.98 0.00

es 0.08 081 0.12 0.02 0.98 0.00

Pro-ß me 0.40 0 53 0.07 0.48 0.52 0.00

es 0.41 0.52 0.07 0.50 0.50 0 00

Pro-Y me 0.07 0.77 0.16 0.01 0.94 0.05

es 0.08 0.77 0.15 0.02 0.94 0.04

Thr-ß me 0.19 0.67 0.14 0.18 0.77 0.05

es 0.19 0.65 0.16 0.18 0.75 0.07

Tyr-8X me 0.14 0.74 0.12 0.11 0.88 0.01

cs 0.15 0.74 0.11 0.12 0.87 0.01

Tyr-ex me 0.28 0.25 0.47 0.30 0.22 0.48

es 0.29 0.24 0.47 0.31 0.21 0.48

93

5.2. Effects of deuterium oxide on the

central carbon metabolism of Escherichia coli

Table 5.2: Relative abundance of intact C2 and C3 fragments in the amino acids from

cultivations in M9 minimal media with 0 % D20, 30 % D20, 50 % D20 and

70 % D20

The table contains /values of those deuterium isotopomers of which the! ^C fine structures could

be sufficiently resolved to assess the relative multiplet intensities. Values at 70 % D20 represent

average values from cultivations with both adapted and non-adapted cells.

Precursor/

D-Isotopomer

M9-

0%D20

Amino

acid

(mediawithD20)

f(i)p2)

Al

)f'D

Pyruvate

ALA-a

CaH--Cf>HD2

CaH-CpD?

averageALA-a

0.01

0.04

0.03

0.92

ALA-ß

CpH3-CuH

CpH3-CaD

CRHoD-CaH

CpHoD-C^D

CpHD2-C(/H

CfiHDv-C(,D

averageALA-ß

0.05

0.95

VAL-a

C„H-Cf,H009

0.00

090

0.01

VAL-Yi

0.05

0.95

LEU-5,

(0.14

0.86)

ILE-Y2

005

0.95

M9

-30%D30

yd)f(2)

fT-)f'Y)

M9

-50%

D-,0

iw

ri]

ri}

.r>

M9

-70%D20

AD

p-2)f(V)

f(3)

0.04

0.06

0.03

0.87(CpH2Dj

0.04

0.07

005

0.84

0.04

0.06

0.02

0.88(CßH3)

0.02

0.09

0.04

0.85

0.04

0.06

0.03

0.87

0.05

0.95

0.07

093

0.06

0.94

006

094

0.06

0.94

0.1

J0.00

0.89

000

0.03

0.08

0.05

084

0.05

095

0.06

0.94

0.05

0.95

008

092

004

096

0.06

0.94

0.10

000

090

000

0.02

0t0

0.03

0.85

0.03

008

0.02

0.87

0.03

0.09

0.02

0.86

0.06

094

0.06

094

0.05

095

005

0OS

005

095

0.07

093

0.06

0.94

0.12

000

088

0.00

Precursor/

D-Isotopomer

M9-

0%D20

Amino

acid

Onedu

withD20)

AD

C2)f'2)

çO)Ribose-5-phosphate

HIS-a

C„H-CßHD

001

002

004

093

HIS-ß

CßHD-CaH

008

062

000

030

H1S-8,

C^H-Cj,009

091

Erythrose-4-pho<>phate

TYR

fCrH

-Cj,("not

.Ksijnt(l)

(1finesliuuureofasuvui)

020

034

046

TYR-8

QH-QH

000

100

000

Phosphoenolpyruvate

PHE-ü

C0H-CpH2

002

008

002

088

PHB-ß

CpHD-C(/U000

100

000

000

TYR-c/

CVH-CpH;

005

007

002

086

TYR-ß

C(jHD-r(,H00!

099

000

000

avciaeePHE-a

TYR-c/

001

008

002

087

l

M9

-30%D20

yO)f'2)

f(2)

yM;

003

001

004

092

009

064

000

027

008

092

020

032

048

000

I00

000

002

008

001

089

003

008

002

087

002

008

002

088

M9

-50

9cD20

fD

f<2,A2

)AV

001

004

005

090

008

064

000

028

007

09"

020

0"2

048

000

I00

000

004

005

003

088

004

007

002

087

004

006

00^

087

M9

-70%D20

f(\>f'2)

pi)

j-n)

002

004

005

089

009

064

000

027

007

093

019

032

049

000

j00

000

004

006

002

088

003

006

002

089

001

006

002

089

Precursor/

amino

acid

D-Isotopomer

(mediawithD,Oi

2-Oxoglutarate(Cj-C^-Cj)

GLU-a

PRO-a

GLU-ß

PRO-ß

ARG-ß

CaH-CßHD

CaH-CßD2

averageGLU-a

CaH-CßHD

CaH-CßD2

M9

-0%D^O

yO)y(2)

/(21fO)

0.24

0.29

0.41

0.06

0.23

0.29

0.42

0.06

averagePRO-u

averageGLU-a.PRO-a

024

028

0.42

006

064

036

000

(055

042

0.03)

0.65

0.35

0.00

so

M9

-30%D20

yd)y'2)

f(T)A3)

M9

-50%D,ö

AD

A.2)

Ol

)AS)

M9

-70%D20

f>»y-2»

f.T)y

(3)

0.23

0.33

0.37

0.07

0.20

0.38

0.38

0.05

0.23

0.32

0.37

0.08(CßH2)

0.20

0.34

0.38

008

0.23

0.32

0.37

0.08

0.20

0.36

0.38

006

0.19

0.35

0.40

004

0.23

0.31

0.40

0.06(CpH2)

0.19

039

039

0.03

023

0.31

0.39

0.07

0.19

037

040

004

020

036

039

005

0.15

0.42

034

0.09

0.16

039

035

0.10

0.15

04)

0.35

0.09

0.15

041

034

0.10

016

040

0.35

009

0.15

041

035

0.09

0.15

041

0.35

0.09

Precursor/

D-Isotopomer

Amino

acid

(mediawithD2Oj

Acetyl-CoA

LEU-cc

CaH-CpHD

2-Oxoglutarate(C4-C5)

ARG-5

C6H2-CyHD

CgH2-CvD2

C8HD-CYH2

C5HD-C7HD

C5HD-CYD2

averageARG-8

PRO-8

PRO-Y

GLU-Y

M9

-0%D20

yCO

y(2)

AT)

y(?)

0.05

0.00

0.95

0.00

0.07

0.93

0.05

095

0.02

091

007

005

000

0.95

0.00

M9

-30%D,0

M9

-50%D70

M9

-70%D,0

yd)y<2)

y(A)

ç(3)fd)

f<2)yG-)

y(3)fO)

y(2)

y(2-)A3)

0.05

0.00

0.95

0.00

0.07

0.00

0.93

000

0.05

0.01

0.94

0.00

0.07

0.93(CYH2)

0.04

0.96

0.05

095

0.05

095

0.04

096

0.06

0.94

0.04

096

0.03

097

0.04

096

0.06

094

0.05

095

004

096

Appendix

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Curriculum vitae

Personal:

Name:

Date of birth:

Place of birth:

Citizenship:

Marital status:

Michel Hochuli

October 19. 1971

Bern, Switzerland

Reitnau (AG), Switzerland

single

Iducaition:

1978--1982

1982 --1986

1986--1990

1991-- 1995

1994--1995

1995 -- 1996

1996 - 1999

Primarschule Bern

Sekundärschule und Untergymnasium Bern

Gymnasium Bern Kirchenfeld. Typus B

Undergraduate studies in chemistry at the University of Bern

emphasis: biochemistry

Student exchange semester at the University of Geneva with

UNIMOBIL

Diploma thesis at the Department of Chemistry and Biochemistry at the

University of Bern (Prof. B. Emi)

subject: The munnose transporter of Escherichia coli: Monitoring

conformational changes in the IIAB subunit by fluorescence

spectroscopy

degree: Dipl. Chem.

Ph. D. thesis at the Institute for Molecular Biology and Biophysics at the

ETH Zürich (Prof. K. Wüthrich & Prof. T. Szyperski)

subject: Metabolic studies of microorganisms using fractional C-

labeling and 2D NMR

degree: Dr. sc. nat. ETH

1998 First year exam at the University of Zürich medical school

^ v£»J fas^ Î

103

Publications

Hoclmli, M.. H. Patzclt, D. Oesterhclt, K. Wüthrich, and T. Szyperski (1999). Amino

acid biosynthesis in the halophilic archaeon Haloarcula hispanica. J. Bacteriol, 181,

3226-3237.

Hochuli, M., T. Szyperski, and K. Wüthrich (1999). D20 isotope effects on the central

carbon metabolism of Escherichia coli cells grown on a minimal medium. In

preparation.

Szyperski, T., R. W. Glaser, M. Hochuli, J. Fiaux, U. Sauer, J. E. Bailey, and K.

Wüthrich (1999). Bioreaction network topology and metabolic flux ratio analysis by1 o

biosynthetic fractional C labeling and two-dimensional NMR spectroscopy.

Metabolic Engineering, 1, 189-197.

Sauer, LT, D. R. Lasko, J. Fiaux, M. Hochuli, R. Glaser, T. Szyperski, K. Wüthrich, and

3. E. Bailey (1999). Metabolic flux ratio (METAFOR) analysis of genetic and

environmental modulations of Escherichia coli central carbon metabolism. J.

Bacteriol., accepted for publication.

Sauer, IT, V. Hatzimanikatis, J. E. Bailey, M. Hochuli, T. Szyperski, and K. Wüthrich

(1997). Metabolic fluxes in riboflavin-producing Bacillus subtilis. Nature

Biotechnol., 15, 448-452.