Evolution & organisation of metabolic pathways

Bas KooijmanDept of Theoretical Biology

Vrije Universiteit, Amsterdamhttp://www.bio.vu.nl/thb/deb/

Amsterdam, 2004/03/31the dynamic structure of life

adul

t

embryo

juvenile

Dynamic Energy Budgettheory for

metabolic organisation

Central Metabolism

polymers

monomers

waste/source

source

• Pentose Phosphate (PP) cycle glucose-6-P ribulose-6-P, NADP NADPH• Glycolysis glucose-6-P pyruvate ADP + P ATP • TriCarboxcyl Acid (TCA) cycle pyruvate CO2

NADP NADPH• Respiratory chain NADPH + O2 NADP + H2O ADP + P ATP

Modules of central metabolism

Evolution of central metabolism

i = inverseACS = acetyl-CoA Synthase pathway PP = Pentose Phosphate cycleTCA = TriCarboxylic Acid cycle

RC = Respiratory Chain Gly = Glycolysis

Kooijman, Hengeveld 2003 The symbiontic nature of metabolic evolution Acta Biotheoretica (to appear)

in prokaryotes (= bacteria)3.8 Ga 2.7 Ga

Prokaryotic metabolic evolution

Chemolithotrophy • acetyl-CoA pathway• inverse TCA cycle• inverse glycolysis

Phototrophy:• el. transport chain• PS I & PS II• Calvin cycle

Heterotrophy:• pentose phosph cycle• glycolysis• respiration chain

Early ATP generationFeS2

FeS

H2

2H+

H2SS0

S0

H2S

2OH-

2H+

ADPATP

Pi2e-

2H2O

FeS + S0 FeS2

ADP + Pi ATP• ATPase• hydrogenase• S-reductase

Madigan et al 1997

Synthesizing Units:generalized enzymesprocess arriving fluxes of substratereversed flux is smallmixtures of processing schemes are possible

Substrate processingFractions of SU·· unbound A· SU-A complex ·B SU-B complexAB SU-A,B complex

Kooijman, 2001

Biomass: reserve(s) + structure(s)Reserve(s), structure(s): generalized compounds, mixtures of proteins, lipids, carbohydrates: fixed composition

Reserve(s) do complicate model & implications & testingReasons to delineate reserve, distinct from structure• metabolic memory• biomass composition depends on growth rate• explanation of respiration patterns (freshly laid eggs don’t respire) method of indirect calorimetry fluxes are linear sums of assimilation, dissipation and growth inter-species body size scaling relationships• fate of metabolites (e.g. conversion into energy vs buiding blocks)

Reserve vs structure

Reserve does not mean: “set apart for later use” compounds in reserve can have active functions

Life span of compounds in• reserve: limited due to turnover of reserve all reserve compounds have the same mean life span

• structure: controlled by somatic maintenance structure compounds can differ in mean life span

Important difference between reserve and structure: no maintenance costs for reserveEmpirical evidence: freshly laid eggs consist of reserve and do not respire

Homeostasis

Homeostasis: constant body composition in varying environments

Strong homeostasis generalized compounds applies to reserve(s) and structure(s) separately

Weak homeostasis: ratio reserve/structure becomes and remains constant if food or substrate is constant (while the individual is growing) applies to juvenile and adult stages, not to embryos

Implication: stoichiometric constraints on growth

DEB decomposition into• assimilation (substrate reserve) catabolic & anabolic aspect• maintenance (reserve products)• growth (reserve structure) catabolic & anabolic aspect

yield coefficients vary with growthreserve, structure differ in compositioncomposition of biomass varies with growth

Methanotrophs

Kooijman, Andersen & Kooi 2004

Macro-chemical reaction at fixed growth rate

OHYNOCHYNHYCOYCH

HXnnnCX

NXCX

NWOWHW 2

324

)1(

CO2

NH3

CH4

O2

reserve

DEB decomposition into• assimilation (substrate reserve) catabolic & anabolic aspect• maintenance (reserve products)• growth (reserve structure) catabolic & anabolic aspect

yield coefficients vary with growthreserve, structure differ in compositioncomposition of biomass varies with growth rm = 0.003 h-1; kE = 0.0127 h-1; kM = 0.0008 h-1

ySE = 8.8; yVE = 0.8 nHE = 2; nOE = 0.46; nNE = 0.25 nHV = 2; nOV = 0.51; nNV = 0.125

Anammox

Brandt, 2002

Macro-chemical reaction at r = 0.0014 h-1

OHNOCHNON

HHCONONH

215.05.0232

324

030.2068.0260.0025.1

128.0068.032.11

Nitrogen cycle

CHON= biomass

some cyanobacteria, Azotobacter, Azospirillum, Azorhizobium, Klebsiella, Rhizobium,some others

Brocadia anammoxidans

Nitrosomonas

Nitrobacter

many

Some crucial conversions depend on few species

SyntrophyCoupling hydrogen & methane production energy generation aspect at aerobic/anaerobic interface

HOHCCHOCOOHC

OHCHHCHOH

HHOHCOOHOHC

222

34

42222

33242262

2432

23322262

ethanol acetate dihydrogen

dihydrogen methane

methane hydrates >300 m deep, < 8Clinked with nutrient supply

bicarbonate

Total:

Product Formation

throughput rate, h-1

glyc

erol

, eth

anol

, g/l

pyru

vate

, mg/

l

glycerol

ethanol

pyru

vate

Glucose-limited growth of SaccharomycesData from Schatzmann, 1975

According to Dynamic Energy Budget theory:

Product formation rate = wA . Assimilation rate + wM . Maintenance rate + wG . Growth rate

For pyruvate: wG<0

Applies to all products, heat & non-limiting substrates

Indirect calorimetry (Lavoisier, 1780): heat = wO JO + wC JC + wN JN

No reserve: 2-dim basis for product formation

Symbiosis

product

substrate

Symbiosis

substrate substrate

Internalization

Structures merge Reserves merge

Free-living, clusteringFree-living, homogeneous

Steps in symbiogenesis

throughput rate

Chemostat Steady Statesbi

omas

s de

nsit

y

hostsymbiont

Free livingProducts substitutable

Free livingProducts complementary

EndosymbiosisExchange on conc-basis

Exchange on flux-basis Structures merged Reserves mergedHost uses 2 substrates

Symbiogenesis

• symbioses: fundamental organization of life based on syntrophy ranges from weak to strong interactions; basis of biodiversity• symbiogenesis: evolution of eukaryotes (mitochondria, plastids)• DEB model is closed under symbiogenesis: it is possible to model symbiogenesis of two initially independently living populations that follow the DEB rules by incremental changes of parameter values such that a single population emerges that again follows the DEB rules• essential property for models that apply to all organisms

Kooijman, Auger, Poggiale, Kooi 2003 Quantitative steps in symbiogenesis and the evolution of homeostasisBiological Reviews 78: 435 - 463

Symbiogenesis1.5-2 Ga 1.2 Ga

Eukaryote metabolic evolution

First eukaryotes: heterotrophs by symbiogenesis compartmental cellular organisationAcquisition of phototrophy frequently did not result in loss of heterotrophyAcquisition of membrane transport between internalization of mitochondria and plastids

No phagocytosis in fungi & plants; loss? pinocytosis in animals = phagocytosis in e.g. amoeba?Direct link between phagocytosis and membrane transport?

Membrane traffic

From: Duve, C. de 1984 A guided tour of the living cell, Sci. Am. Lib., New York

The golgi apparatus servesas a central clearing houseand channel between the endo- and exoplasmic domains

1 ER-Golgi shuttle2 secretory shuttle between Golgi and plasma membrane2’ crinophagic diversion3 Golgi-lysosome shuttle3’ alternative route from Golgi to lyosomes via the plasma membrane and an endosome4 endocytic shuttle between the plasma membrane and an endosome4’ alternative endocytic pathway bypassing an endosome5 plasma membrane retrieval6 endosome-lysosome pathway7 autophagic segregation

Clathrin unknown in prokaryotes

Chloroplast dynamics

Coordinated movement of chloroplasts through cells

Bacillariophyceae(diatoms)

(brown algae)Phaeophyceae

Prymnesiophyceae

RaphidophyceaeXanthophyceae

EustigmatophyceaeDictyochophyceae

Pelagophyceae

ChrysophyceaeSynurophyceae

Cryptophyceae

(plants)Cormophyta

(green algae)Chlorophyceae

(red algae)Rhodophyceae

Glaucophyceae

animals

Euglenozoa

Dinozoa

Rhizopoda

Bicosoecia

Actinopoda

Pseudofungi

Labyrinthulomycota

MyxomycotaProtostelida Ciliophora

Sporozoa

Bacteria

Zygomycota

BasidiomycotaAscomycota

Archamoeba

Microsporidia

Chytridiomycota

Percolozoa

Bigyromonada

Metamonada

Choanozoa

GranuloreticulataXenophyophora

Loukozoa

PlasmodiophoromycotaChlorarachnida

Cercomonada

Apusozoa

Pedinellophyceae

Bolidophyceae

Composed byBas Kooijman

Opalinata

Glomeromycota

Survey of organisms

mitochondria

secondarychloroplast

primary chloroplast

tertiarychloroplast

Sizes of blobsdo not reflect

number of species

Bacteria

Opi

stho

kont

s

Chromista

Amoebozoa

Alveo-lates

Plantae

Excavates

Ret

aria

Cercozoa

fungi

animals

forams

cort

ical

alv

eoli

Bik

ont

DH

FR

-TS

gen

e fu

sion

chlo

ropl

asts

mem

br. d

ynun

ikon

t

loss phagoc.gap junctions tissues (nervous)

bicentriolarmainly chitin

EF1 insertion

trip

le r

oots

mai

nly

cell

lose

photosymbionts

Cells, individuals, colonies

• plasmodesmata connect cytoplasm; cells form a symplast: plants

• pits and large pores connect cytoplasm: fungi, rhodophytes

• multinucleated cells occur; individuals can be unicellular: fungi, Eumycetozoa, Myxozoa, ciliates, Xenophyophores, Actinophryids, Biomyxa, diplomonads, Gymnosphaerida, haplosporids, Microsporidia, nephridiophagids, Nucleariidae, plasmodiophorids, Pseudospora, Xanthophyta (e.g. Vaucheria), most classes of Chlorophyta (Chlorophyceae, Ulvophyceae, Charophyceae (in mature cells) and all Cladophoryceae, Bryopsidophyceae and Dasycladophyceae)) • cells inside cells: Paramyxea • uni- and multicellular stages: multicellular spores in unicellular myxozoa, gametes• individuals can remain connected after vegetative propagation: plants, corals, bryozoans• individuals in colonies can strongly interact and specialize for particular tasks: syphonophorans, insects, mole rats

vague boundaries

Kooijman, Hengeveld 2003 The symbiontic nature of metabolic evolution Acta Biotheoretica (to appear)

rotiferConochilus hippocrepisHeterocephalus glaber

(Endo)symbiosisFrequent association between photo- and heterotroph photo hetero: carbohydrates (energy supply) photo hetero: nutrients (frequently NH3 or NO3

-) most (perhaps all) plants have myccorrhizas, the symbiosis combines photolithotrophy and organochemotrophy

Also frequent: association between phototroph and N2-fixer where N2-fixer plays role of heterotroph

Symbiosis: living together in interaction (basic form of life)Mutualism: “benefit” for both partners symbioses need not be mutualistic “benefit” frequently difficult to judge and anthropocentricSyntrophy: one lives of products of another (e.g. faeces) can be bilateral; frequent basis of symbiosis

Chlorochromatium (Chlorobibacteria, Sphingobacteria)

From: Margulis, L & Schwartz, K.V.1998 Five kingdoms.Freeman, NY

(= Chlorochromatium)

(Endo)symbiosisParamecium bursariaParamecium bursariaciliate with green algaeciliate with green algae

Ophrydium versatileOphrydium versatileciliate with green algaeciliate with green algae

PeltigeraPeltigeraascomycete with green algaeascomycete with green algae

Cladonia diversaCladonia diversaascomycete with green algaeascomycete with green algae

(Endo)symbiosis

Chlorophyte symbiontsChlorophyte symbiontsvisible through microscopevisible through microscope

Lichen Lichen Cladonia portentosaCladonia portentosa

Grazed by reindeer in winterGrazed by reindeer in winterRangifer tarandusRangifer tarandus

Mitochondria

Transformations:1 Oxaloacetate + Acetyl CoA + H2O = Citrate + HSCoA2 Citrate = cis-Aconitrate + H2O3 cis-Aconitrate + H2O = Isocitrate4 Isocitrate + NAD+ = α-Ketoglutarate + CO2 + NADH + H+

5 α-Ketoglutarate + NAD+ + HSCoA = Succinyl CoA + CO2 + NADH + H+

6 Succinyl CoA + GDP 3- + Pi 2- + H+ = Succinate + GTP 4- + HSCoA

7 Succinate + FAD = Fumarate + FADH2

8 Fumarate + H2O = Malate9 Malate + NAD+ = Oxaloacetate + NADH + H+

TriCarboxylic Acid cycle (= Krebs cycle)

Enzymes pass metabolites directly to other enzymes enzymes catalizing transformations 5 & 7: bound to inner membrane (and FAD/FADH2)Net transformation: Acetyl-CoA + 3 NAD+ + FAD + GDP 3- + Pi

2- + 2 H2O = 2 CO2 + 3 NADH + FADH2 + GTP 4- + 2 H+ + HS-CoA

Dual function of intermediary metabolites building blocks energy substrate all eukaryotes

once possessed mitochondria,most still do

enzymes are located in metabolon;channeling of metabolites

Pathways & allocation

reserve

reservereserve

maintenance

maintenance

maintenance

structure structure

structure

Mixture of products &intermediary metabolites

that is allocated tomaintenance (or growth)has constant composition

Kooijman & Segel, 2004

Numerical matching for n=4P

rodu

ct f

lux

Rej

ecte

d fl

ux

Unb

ound

fra

ctio

n

= 0.73, 0.67, 0.001, 0.27 handshaking = 0.67, 0.91, 0.96, 0.97 binding probk = 0.12, 0.19, 0.54, 0.19 dissociation nSE = 0.032,0.032,0.032,0.032 # in reservenSV = 0.045,0.045,0.045,0.045 # in structureyEV = 1.2 res/struct kE = 0.4 res turnover jEM = 0.02 maint flux n0E = 0.05 sub in res

0

0

1

1

1

2

2

23

3

3

4

4

Spec growth rate

Spec growth rate

Matching pathway whole cellNo exact match possible between production of products and intermediary metabolites by pathway and requirements by the cell

But very close approximation is possible by tuning abundance parameters and/or binding and handshaking parameters

Good approximation requires all four tuning parameters per node growth-dependent reserve abundance plays a key role in tuning

VSES iinn ,

ii αρ ,

Kooijman, S. A. L. M. and Segel, L. A. (2004) How growth affects the fate of cellular substrates.Bull. Math. Biol. (to appear)