xyloglucan-active enzymes: properties, structures and - diva portal
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Xyloglucan-active enzymes: properties,
structures and applications.
Martin J. Baumann Doctoral thesis
School of Biotechnology
Royal Institute of Technology
Stockholm 2007
ii
Picture on front page shows two xyloglucan oligosaccharides, as they were observed in the active site of CtXGH74A (PDB code 2CN3), an inverting GH74 xyloglucan hydrolase (Paper 1, page 34). Two xyloglucan oligosaccharides were observed, left: XLLG (short hand notation of Fry et al., 1993), right: XXLG. The glucose backbone is depicted in yellow; xyloses are shown in light green and galactose residues in orange. The catalytic residues of CtXGH74A are shown in green; D70 is in front and D480 behind the oligosaccharides. Picture is arranged and rendered with PyMOL (DeLano, W.L. The PyMOL Molecular Graphics System (2002) DeLano Scientific, Palo Alto, CA, USA. http://www.pymol.org)
© Martin J. Baumann
School of Biotechnology
Royal Institute of Technology
AlbaNova University Center
106 91 Sweden
ISBN 978-91-7178-596-1
Abstract
iii
Cellulosic materials are the most abundant renewable resource in the world; plant cell walls are natural composite materials containing crystalline cellulose embedded in a matrix of hemicelluloses, structural proteins, and lignin. Xyloglucans are an important group of hemicelluloses, which coat and cross-link crystalline cellulose in the plant cell wall. In this thesis, structure-function relationships of a range of xyloglucan-active enzymes were examined.
A paradigm for efficient enzymatic biomass utilization is the cellulosome of the anaerobic bacterium Clostridium thermocellum. The cellulosome is a high molecular weight complex of proteins with diverse enzyme activities, including the inverting xyloglucan endo-hydrolase CtXGH74A. The protein structure of CtXGH74A was solved in complex with xyloglucan oligosaccharides (XGOs) which stabilized disordered loops of the apo-structure. Further detailed kinetic and product analyses were used to conclusively demonstrate that CtXGH74A is an endo-xyloglucase that produces Glc4-based XGOs as limit digestion products.
In comparison, the retaining glycoside hydrolase family 16 (GH16) contains hydrolytic endo-xyloglucanases as well as xyloglucan transglycosylases (XETs) from plants. To elucidate the determinants of the transglycosylase/hydrolysis ratio in GH16 xyloglucan-active enzymes, a strict transglycosylase, PttXET16-34 from hybrid aspen, was compared structurally and kinetically with the closely related hydrolytic enzyme NXG1 from nasturtium. A key loop extension was identified in NXG1, truncation of which yielded a mutant enzyme that exhibited an increased transglycosylase rate and reduced hydrolytic activity. Kinetic studies were facilitated by the development of new, sensitive assays using well-defined XGOs and a series of chromogenic XGO aryl-glycosides.
A detailed understanding of GH16 xyloglucan enzymology has paved the way for the development of a novel chemo-enzymatic approach for biomimetic fiber surface modification, in which the transglycosylating activity of PttXET16-34 was employed. Aminoalditol derivates of XGOs were used as key intermediates to incorporate novel chemical functionality into xyloglucan, including chromophores, reactive groups, protein ligands, and initiators for polymerization reactions. The resulting modified xyloglucans were subsequently bound to a range of cellulose materials to radically alter surface properties. As such, the technology provides a novel, versatile toolkit for fiber surface modification.
Sammanfattning
iv
Cellulosabaserade material är världens rikligast förekommande
förnyelsebara råvara. Växters cellväggar är naturliga kompositmaterial
där den kristallina cellulosan är inbäddad i en väv av hemicellulosa,
strukturproteiner och lignin. Xyloglukaner är en viktig
hemicellulosagrupp som omger och korslänkar den kristallina cellulosan i
cellväggarna. I denna avhandling undersöks undersöks sambanden
mellan struktur och funktion hos olika xyloglukan-aktiva enzymer.
En modell för effektiv enzymatisk omvandling av biomassa ges av
cellulosomen hos den anaeroba prokaryota organismen Clostridium
thermocellum. Cellulosomen är ett proteinkomplex med hög molmassa
och flera olika enzymaktiviteter, bl.a. det inverterande xyloglukan-
endohydrolaset CtXGH74A. Proteinstrukturen för CtXGH74A har lösts i
komplex med xyloglukanoligosackarider, som stabliliserar vissa
loopar/slingor som är oordnade i apostrukturen. Ytterligare detaljerade
kinetiska och produktananalyser har genomförts för att entydigt visa att
CtXGH74A är ett endoxyloglukanas vars slutliga nedbrytningsprodukt är
Glc4-baserade xyloglukanoligosackarider.
Som jämförelse innehåller glykosidhydrolasfamilj 16 (GH16) såväl
hydrolytiska endoxyloglukanaser som xyloglukantransglykosylaser
(XETs) från växter. För att utreda vad som bestämmer förhållandet
mellan transglykosylering och hydrolys i xyloglukanaktiva enzymer från
familj GH 16 jämfördes struktur och kinetik hos ett strikt
transglykosylas, PttXET16-34 från hybridasp, med ett nära besläktat
hydrolytiskt enzym, NXG1 från krasse. I NXG1 identifierades en viktig
förlängningsloop, som vid trunkering gav ett muterat enzym med högre
transglykosyleringshastighet och minskad hydrolytisk aktivitet.
Kinetikstudierna genomfördes med hjälp av nyutvecklade känsliga
provmetoder med väldefinerade XGO:er och ett antal kromogena XGO-
arylglykosider.
En detaljerad förståelse av enzymologin inom GH16 möjliggjorde
utvecklingen av en ny kemoenzymatisk metod för biomimetisk
fiberytmodifiering med hjälp av PttXET16-34s
translgykosyleringsaktivitet. Aminoalditolderivat av
xyloglukanoligosackarider användes som nyckelintermediärer för att
introducera ny kemisk funktionalitet hos xyloglukan, såsom kromoforer,
reaktiva grupper, proteinligander och initiatorer för
polymeriseringsreaktioner. Tekniken innebär ett nytt och mångsidigt
verktyg för fiberytmodifiering.
Zusammenfassung
v
Zellulosehaltige Materialien sind die häufigsten erneuerbaren
Rohmaterialien auf der Welt. Pflanzenzellwände sind natürliche
Kompositmaterialien, sie enthalten kristalline Zellulose, die in einer Matrix aus
Hemizellulosen, Proteinen und Lignin eingebettet sind. Xyloglukane sind eine
wichtige Gruppe der Hemizellulosen, sie ummanteln und verbinden Zellulose in
der pflanzlichen Zellwand. In dieser Abhandlung werden Strukturen von drei
Xyloglukanaktiven Enzymen in Beziehung zu ihrer Funktion untersucht.
Ein Paradigma für effizienter Nutzung von Biomasse ist das Cellulosom
des anaerob lebenden Bakteriums Clostridium thermocellum. Das Cellulosom
ist ein hochmolekularer Komplex von Proteinen mit vielen verschiedenen
Aktivitäten, darunter ist auch die invertierende Xyloglukan Endohydrolase
CtXGH74A. Die Proteinstruktur von CtXGH74A wurde im Komplex mit
Xyloglukanoligosacchariden (XGO) gelöst, welche ungeordnete Loops der apo-
Struktur stabilisierten. Durch weitere detaillierte Analyse der Kinetik und
Reaktionsprodukte konnte schlüssig gezeigt werden, daß CtXGH74A eine
Endoglukanase ist, die Glc4-basierte XGO produziert.
Im Vergleich dazu enthält die retentierende Glykosidhydrolasefamilie 16
(GH16) sowohl hydrolytische Endoxyloglukanasen als auch Transglykosidasen
von Pflanzen. Um zu erklären welche Faktoren das Verhältnis zwischen
Transglykosidase und Hydrolase Aktivität bei GH16 Xyloglukanaktiven Enzymen
bestimmen wurde eine reine Transglykosidase PttXET16-34 von Hybridaspen
mit einem nah verwandten hydrolytischen Enzym NXG1 von Kapuzinerkresse
strukturell und kinetisch verglichen. Als Schlüsselstelle wurde eine
Verlängerung eines Loops in NXG1 identifiziert, Verkürzung des Loops führte zu
einer Mutante mit erhöhter Transglykosylierungsrate bei verminderter
hydrolytischer Aktivität. Kinetische Studien wurden erleichtert durch neu
entwickelte hochempfindliche Methoden für Aktivitätsmessung, die auf XGO
oder chromogene Aryl-XGO als definierte Substrate zurückgreifen.
Detailliertes Verständnis von GH16 Enzymologie hat den Weg für die
Entwicklung für eine neuartige Methode für biomimetische
Oberflächenmodifikation von Zellulosefibern geebnet, dafür wurde die
transglykosylierende Aktivität von PttXET16-34 angewendet. Aminoalditol-
derivate von XGO wurden als wichtigste Zwischenprodukte angewendet, um
neue chemische Funktionalitäten in Xyloglukan einzuführen, darunter waren
Chromophore, reaktive Gruppen, Proteinliganden und Initiatoren für
Polymerisationsreaktionen. Die modifizierten Xyloglukane wurden an eine
Reihe von verschiedenen Zellulosematerialien gebunden und veränderten die
Oberflächeneigenschaften dramatisch. Diese Methode ist ein neues wertvolles
Werkzeug für Oberflächenmodifikation von Zellulosen.
vi
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List of publications 1 Martinez-Fleites* C., Guerreiro* C. I. P. D., Baumann* M.
J., Taylor E. J., Prates J. A. M., Ferreira L. M. A., Fontes C. M. G. A., Brumer H. and Davies G. J. (2006). "Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation." Journal of Biological Chemistry; 281(34), 24922-24933. (* authors share first name)
2 Baumann M. J., Eklöf J., Michel G., Å. Kallas, Teeri T. T.,
Czjzek M., Brumer H. (2007). Structural analysis of nasturtium NXG reveals the evolution of GH16 xyloglucanase activity from XETs: biological implications for cell wall metabolism. Manuscript.
3 Johansson P,. Brumer H. III, Baumann M. J., Kallas Å.,
Henriksson H., Denman S., Teeri T. T. and Jones A. (2004). Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of the transglycosylation acceptor binding. Plant Cell (16), 874-886
4 Kallas, Å. M., Baumann M. J., Fäldt J., Aspeborg H., Denman S. E., Mellerowicz E. J., Nishikubo N., Brumer H. and Teeri T. T. (2006). "Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloides." Manuscript.
5 Brumer, H. III, Zhou Q., Baumann M. J., Carlsson, K. and
Teeri T. T. (2004). Activation of crystalline cellulose surfaces though the chemoenzymatic modification of xyloglucan. (2004) Journal of the American Chemical Society; 126 (18), 5715-5721
viii
Additional publications: Zhou, Q., Baumann M. J., Brumer H. and Teeri T. T. (2006). "The influence of surface chemical composition on the adsorption of xyloglucan to chemical and mechanical pulps." Carbohydrate Polymers 63(4): 449-458. Zhou, Q., Baumann M. J., Piispanen P. S., Teeri T. T. and Brumer H. (2006). "Xyloglucan and xyloglucan endo-transglycosylases (XET): Tools for ex vivo cellulose surface modification." Biocatalysis and Biotransformation, 24(1/2): 107-120. Zhou, Q., Greffe L., Baumann M. J., Malmström E., Teeri T. T. and Brumer H. (2005). "Use of xyloglucan as a molecular anchor for the elaboration of polymers from cellulose surfaces: A general route for the design of biocomposites." Macromolecules 38(9): 3547-3549.
Table of contents
1
1. Introduction............................................................................ 2 1.1. Biotechnology in the pulp and paper industry ................................ 3 1.2. Understanding and applying enzymes .......................................... 5
2. Xyloglucan is a cell wall polysaccharide.................................. 6 2.1. Function of xyloglucans in plants ................................................. 7 2.1.1. Xyloglucan as a hemicellulose in the cell wall ............................. 7 2.1.2. Xyloglucan as storage polysaccharide ....................................... 8
2.2. Structure of xyloglucan .............................................................. 9
3. Material properties of xyloglucan.......................................... 12 3.1. Xyloglucan sources.................................................................. 12 3.2. Xyloglucan solution properties and applications ........................... 13 3.3. Interaction of xyloglucan and cellulose ....................................... 15
4. Carbohydrate active enzymes in the biosynthesis and degradation of xyloglucan ........................................................ 18
4.1. Enzyme classification ............................................................... 18 4.1.1. Enzyme nomenclature according to NC-IUBMB ......................... 18 4.1.2. Carbohydrate-Active enzymes ............................................... 21
4.2. Xyloglucan biosynthesis in the plant cell ..................................... 21 4.3. GH hydrolases ........................................................................ 23 4.4. Inverting xyloglucan-hydrolases ................................................ 23 4.4.1. Inverting mechanism............................................................ 24 4.4.2. Inverting xyloglucan hydrolases in the partial enzymatic
degradation of plant cells wall by bacteria ............................... 25 4.5. Retaining xyloglucan hydrolases ................................................ 26 4.5.1. Retaining mechanism of GH16 hydrolases ............................... 26 4.5.2. Biological role of GH16 hydrolases in plants ............................. 27
4.6. Retaining GH transglycosylases ................................................. 28 4.6.1. Mechanism of retaining xyloglucan endotransferases................. 28 4.6.2. Retaining transglycosylases among GH enzymes ...................... 29 4.6.3. Biological roles of GH16 enzymes in cell expansion ................... 31
5. Aims of the present investigation ......................................... 33
6. Results and discussion.......................................................... 34 6.1. CtXGH74A structure and properties ........................................... 34 6.2. NXG1 structure and properties .................................................. 36 6.3. PttXET16-34 structure ............................................................. 40 6.3.1. PttXET16-35 characterization................................................. 42
6.4. Fiber surface modification......................................................... 43
7. Concluding remarks and future perspectives ........................ 46
8. Acknowledgements............................................................... 47
9. List of abbreviations ............................................................. 48
10. References ............................................................................ 49
Martin J. Baumann
2
1. Introduction Conversion of biomass into energy and raw materials is the key to
a long term sustainable development. Cellulosic materials are the
most abundant renewable resource in the world. Cellulosic
materials can be used for generating biofuels (bioethanol), or for
light weight composite materials. As the trend is towards more
environmentally friendly materials, interest in renewable fibers as
the reinforcing component has been stimulated.
The performance of biomaterials depends on a detailed
understanding of their composition and structure. An invaluable
way to understand natural materials is to study their assembly
and degeneration in nature. By studying the enzymatic machinery
which builds up or degrades cellulosic materials we can learn how
to utilize these materials to our greater advantage. Basic
biochemical studies are the foundation for the use of enzymes in
any application. Biochemical characterizations of enzymes
currently represent a bottleneck for the applications of enzymes in
industry.
In nature, cellulose is embedded in a complex mixture of
hemicelluloses and lignin in the secondary cell wall. Xyloglucan
(XG) is one important hemicellulose in a large proportion of the
plant kingdom. Xyloglucan embeds and cross-links the crystalline
cellulose in plant primary cell walls. A special class of enzymes
remodels the xyloglucan in the cell wall, and these non-hydrolytic
enzymes can be used to modify cellulose fibers for high
performance products such as biomaterials1.
1 This PhD project is partly funded by Swedish Forest Industries Federation (Skogsindustrierna) through the Biofibre Material Centre (BiMaC), which is gratefully acknowledged. More information is available at http://www.skogsindustrierna.org and http://www.bimac.kth.se
Introduction
3
1.1. Biotechnology in the pulp and paper industry Forests cover approximately half of Sweden’s land area, and
the forest industry has been one of Sweden’s largest industries for
decades. In 2003, forest products contributed 13 % to Sweden’s
total export value. 2 Both in the Swedish and in a global
perspective, forest products have a significant economic role. In
the early 1980’s the development of modern biotechnology
started, and until now, it has had its biggest impact in the
pharmaceutical area. Already at that time the pulp and paper
industry was well established with large scale production facilities
and heavy and expensive machinery. The processes have been
developed largely without the application of biotechnology, and
the large scale of the current production facilities makes it difficult
to apply biotechnology without modifications of the established
processes.
Despite this, certain advances in the application of
biotechnology to the pulp and paper industry have been made, for
instance in the bleaching process. Lignin reduction is typically
achieved by bleaching the pulp with oxidizing agents. In order to
reduce the consumption of bleaching chemicals there have been
attempts to reduce, or simplify the bleaching process. Different
strategies have been undertaken to reduce the lignin content of
the raw material prior to bleaching. Trees have been modified
genetically (Hu et al., 1999; Sederoff, 1999), or white rot fungi
have been applied on wood chips prior to processing. (Messner et
al., 2003; Wolfaardt et al., 2004) Among the few enzymes used
in bulk in the pulp and paper industry, xylanases are used as a
bleach booster (Harris et al., 1997; Bajpai, 1999; Kim and Paik,
2000; Bajpai, 2004; Dutta et al., 2007). According to the current
2 Statistical Yearbook of Forestry 2006 (available at http://www.skogsstyrelsen.se)
Martin J. Baumann
4
hypothesis, xylanases hydrolyze xylans during enzymatic
bleaching and thereby prevent re-precipitation of xylans onto the
fibers upon removal of bleaching chemicals. Re-precipitation of
xylans can entrap lignin on the fiber surface, glucuronic acids in
xylan contribute to the yellowing of aged paper.
The inter-fiber bonds in the formed paper sheet are of
utmost importance for the product properties. Polymeric additives
can stabilize the inter-fiber bond, such cross-linking agents are
currently made chemically (Xu et al., 2005; Chen et al., 2006;
Wang et al., 2006). In the future, such modifications of the sheet
might be made in a biomimetic way by hemicelluloses, such as
xylan (Westbye et al., 2006) or xyloglucan (Christiernin et al.,
2003; Lima et al., 2003; Whitney et al., 2006).
Slime and pitch control is very important, since the
runability can be negatively effected. Lipases, which are highly
stable enzymes, are widely applied to solubilize hydrophobic
substances. By using lipases, disturbances in the forming paper,
which potentially can lead to breaks, are less frequent (Chaudhary
et al., 1998; Bajpai, 1999).
Regarding composite materials, cellulosic fibers can be used
as a lightweight and stiff reinforcement material. In order to yield
a strong composite material, the interfacial incompatibility
between hydrophobic embedding materials and the hydrophilic
fiber has to be overcome (Trejo-O'Reilly et al., 1997). Fiber
surface modification like the XET-technology (Paper 5, Lönnberg
et al., 2006) can help to overcome this problem, while maintaining
the fiber stiffness.
Research on wood formation in nature will lead to the
discovery of new enzymes, and new insights about the role of cell
wall polymers. This knowledge will lead to innovative
biotechnology for the forest industry.
Introduction
5
1.2. Understanding and applying enzymes High selectivity and mild reaction conditions make enzymes
a very attractive tool for modern, environmentally friendly
applications. Detailed knowledge about enzymes is a prerequisite
for a successful application in biotechnological processes. This
includes fundamental studies on substrate specificity and specific
activities, but also more advanced indicators, such as kinetic
parameters and protein structures. Often, enzyme engineering is
required to change or modify enzyme activities, or stability.
(Igarashi et al., 2003; Reetz et al., 2006) Three-dimensional
structures of enzymes, for instance determined by X-ray
crystallography, are useful tools for understanding the enzymatic
mechanism and the interaction between enzyme and substrate.
Structures with ligands can be difficult to obtain, as the substrate
can be converted by the enzyme activity. The easiest way to
prevent turnover of the substrate is to provide the product of the
reaction catalyzed. Another often practiced technique is to use an
inactive mutant for crystallization (as exemplified in Martinez-
Fleites et al., 2006). A necessary condition for structural studies
is sufficient amount of protein of a high purity. Protein can be
provided by purification from the natural source, or by
heterologous protein expression. When heterologous protein
expression is used, protein variants can be produced by well
known DNA manipulation techniques.
Martin J. Baumann
6
2. Xyloglucan is a cell wall polysaccharide Plant cell walls are built from a mixture of polysaccharides,
proteins and lignin. The non-crystalline matrix of plant cell walls
is built of one or more of the different types of hemicelluloses such
as galactoglucomannan, glucomannan, arabinoglucoronoxylan,
glucuronoxylan and xyloglucan (Timell, 1967). Hemicellulose is an
old-fashioned term which describes the non-pectin, base-
extractable polysaccharide compound of the cell wall. Some
hemicelluloses are more common in primary cell walls, others
occur in the secondary cell wall. The type and composition of
hemicelluloses differ from plant to plant. Hemicelluloses form the
matrix in which the cellulose microfibrils are embedded (Figure 2).
Flowering Plants
Monocotyledons
Dicotyledons
Commelinoid
Non Commelinoid
Cell wallsGlucuronoarabinoxylansFerrulic acid(1→3), (1→4)-β-glucans (grasses)
Storage Fructanβ(2→6)−fructans (grasses)
Cell wallsXyloglucansPectic Polysaccharides
Storage Fructanβ(2→1)−fructans
Figure 1. Composition of the cell wall is species dependent; xyloglucan is the principal hemicellulose of primary cell walls of dicots and in about half of the monocots.
Xyloglucan is a cell wall polysaccharide
7
2.1. Function of xyloglucans in plants Xyloglucans (XGs) are widely distributed throughout the
plant kingdom (Figure 1), in which they play distinct roles as both
structural and storage polysaccharides. XGs have been found in
both monocots and dicots, as well as in certain non-vascular
plants (Kooiman, 1960; Fry, 1989; Hayashi, 1989; Pauly et al.,
1999a; Popper and Fry, 2003; Fry, 2004; Tine et al., 2006).
2.1.1. Xyloglucan as a hemicellulose in the cell wall
Xyloglucan is the principal hemicellulose of primary cell walls
of dicots and in about half of the monocots (Figure 1). Xyloglucan
may contribute to up to one-fourth the dry weight of the primary
cell wall. The evolutionary occurrence of xyloglucan in plants is
believed to coincide with the occurrence of land plants (Fry,
1989). To adjust to the new environment, the cell wall had to be
stiffened for improved load bearing properties. It is thought that
xyloglucan molecules form a sheath around the cellulose
microfibrils preventing them from direct aggregation. Xyloglucan
can even be woven into the amorphous parts of the microfibrils.
Other xyloglucan molecules cross-link to different cellulose
microfibrils, and provide a flexible and strong network in the
primary cell wall (Figure 2).
Martin J. Baumann
8
Figure 2. The xyloglucan polysaccharides in the primary cell wall cover the cellulose microfibrils and connect adjacent microfibrils. Picture adapted from Rose and Bennett (1999).
2.1.2. Xyloglucan as storage polysaccharide
Xyloglucan is the predominant storage polysaccharide in
seed endosperm cell walls of many plants (Kooiman, 1960; Tine et
al., 2006). The presence of xyloglucan in seeds is known since
the 19th century (Edwards et al., 1985); it stains, similar to starch,
green-bluish with iodine and was called Amyloide3. In the context
of this work, two relevant examples of storage xyloglucan are
those from the seeds of the sub-tropical Tamarindus indica
(tamarind) and the nasturtium flower (Tropaloelum majus) (Reid,
1985; Buckeridge et al., 2000). As a storage saccharide,
xyloglucan is deposited in layers onto the cell wall. During
germination, the storage polysaccharide is mobilized by hydrolytic
extra-cellular enzymes (Edwards et al., 1985).
3 The word “amyloid” is presently used for insoluble fibrous protein aggregations. The name amyloid comes from the early mistaken identification of the substance as starch (amylum in Latin), based on crude iodine-staining techniques.
Xyloglucan is a cell wall polysaccharide
9
2.2. Structure of xyloglucan Xyloglucan is structurally related to cellulose as it shares the
same backbone of β(1→4)-linked glucose residues. The observed
chain length of xyloglucan varies from 300 to 3000 glucose units
(Fry, 1989). The typical repeating unit contains four glucose
units. The three most distant glucose residues from the reducing
end are substituted with α(1→6) xylose residues (Figure 3,
Vincken et al., 1996b; Vincken et al., 1997b).
The decoration of xyloglucan differs from plant species to
plant species (Table 2), heterogeneity of xyloglucan has also been
observed between different tissues of the same plant (Hoffman et
al., 2005b). Some xylose units are further substituted by β−D-
Galp-(1→2) or α-L-Fucp(1→2)-β−D-Galp-(1→2). The side chains
O O
HOHO
OH
OO
HOHO
OO
HOHO
O
OO
HO
HO
O
HO
O
HO
O
OH
O
OH
OH
OH
OHO
OH
OH
OH
O
OH
O
HO
O
OHO
OHHO
HO
H
n
y
x
Figure 3. The structure of the repeating unit of most xyloglucans: four β(1→4)-linked glucose units, substituted with three α(1→6)-linked xylose units, which can be substituted with β(1→2)-linked galactose (when x=1 y=1 XLLG) and in some cases further with arabinose or fucose (not shown). A selection of other xyloglucan repeating units is shown in Table 2, the repeating unit is generally identified as XGO.
Martin J. Baumann
10
on xyloglucan dramatically change the physical properties of the
polymer compared to cellulose; xyloglucan is highly water soluble
and cannot form ordered crystalline microfibrils like cellulose (Fry,
1989). The high degree of branching makes the structural
description of on xyloglucan dramatically change the physical
properties of the polymer complicated, therefore a single letter
short hand description (Table 1) is widely used (Fry et al., 1993).
In addition, xyloglucan can be partly acetylated after synthesis in
the Golgi apparatus (Carpita and Gibeaut, 1993).
Table 1. Short hand notation for xyloglucan oligosaccharides after (Fry et al., 1993)
Short hand notation Monosaccharides and linkage
G Glcp
X α-D-Xylp(1→6)-Glcp
L β−D-Galp(1→2)- α-D-Xylp(1→6)-Glcp
F α-L-Fucp(1→2)-β−D-Galp(1→2)- α-D-Xylp(1→6)-Glcp
U β−D-Xylp(1→2)-α-D-Xylp(1→6)-Glcp (Hilz et al., 2007)
S α-D-Araf(1→2)- α-D-Xylp(1→6)-Glcp
Xyloglucan is a cell wall polysaccharide
11
Table 2. Composition of on xyloglucan dramatically change the physical properties of the polymer from selected sources, table modified after (Zhou et al., 2006b) Sources
Xyloglucan oligosaccharides compositions References
Bilberry (Vaccinium myrtillus L)
XXXG, XXUG, XXLG, XXFG, XLFG4 (Hilz et al., 2007)
Black currant, (Ribes nigrum L)
XXXG, XXFG, XLFG, XLXG , XXLG , XLLG (30:31:35:2:2:1)
(Hilz et al., 2006)
Afzelia africana
XXXG, XLXG, XXLG, XLLG (1.00:0.21:2.80:1.68) (Ren et al., 2004)
Tamarindus indica kernel powder
XXXG, XLXG, XXLG, XLLG (1.000:1.160:1.240:1.429)
(Marry et al., 2003)
Hymenaea courbaril cotyledones
XXXXG based subunits and Glc6 based subunits (Buckeridge et al., 1997; Tine et al., 2003; Tine et
al., 2006) Olive (olea europaea cv koroneiki) fruit pulp
XXSG, XLSG, some of the arabinose residues are substituted with either 1 or 2 O-acetyl groups
(Vierhuis et al., 2001)
Persimmon (Diospyros kaki L.) fruit pulp
Glc:Xyl:Gal:Fuc (10.0:6.0:3.4:1.4) (Cutillas-Iturralde et al., 1998)
Apple (Malus malus L.) fruit pulp
XXXG, XLXG, XXFG, XLFG (4:2:3:4) (Vincken et al., 1996a) (Spronk et al., 1997)
Secreted from suspension culture of Nicotiana plumbaginifolia
Based on subunits XXGG (34%) and XXGGG (27%), one (60%) or two (15%) terminal Araf residues attached at O-2 of the Xylp residues.
(Sims et al., 1996)
Potato (Solanum tuberosum var. Bintje)
XXGG-type subunits (Vincken et al., 1996b)
Detarium senegalese Gmelin cotyledons
XXXG, XLXG, XXLG, XLLG (1.00:0.30:5.60:6.20) (Wang et al., 1996)
Tamarindus indica xyloglucan, sycamore extra-cellular polysaccharide (Acer pseudoplatanus) and rapeseed hulls (brassica naptus)
XG from rapeseed: XXXG, XXLG, XLXG, XXFG, XLFG (11.5:1:3:9.4:16.9) XG from tamarind seed: XXXG, XXLG, XLXG, XLLG (1.4:3:1:5.4)
(York et al., 1990; Hisamatsu et al., 1991; Hisamatsu et al., 1992;
York et al., 1993; York et al., 1995)
Suspension-cultured poplar cells
Wall XG: XLFG, XXFG, XXLG, XXXG (0.58:1.76:0.54:1.0) Extra-cellular XG: XLFG, XXFG, XXLG, XXXG (0.53:2.08:0.65:1.0)
(Hayashi and Takeda, 1994)
4 More than 20 oligosaccharides have been identified, only the most abundant (more than 10%) are listed here.
Martin J. Baumann
12
3. Material properties of xyloglucan
3.1. Xyloglucan sources The most commonly used on xyloglucan dramatically change
the physical properties of the polymer is from Tamarindus indica.
Tamarind is a sub-tropical tree; the seeds are surrounded by an
acidic paste, containing oxalic acid. The dried fruit pulp can be
used as a spice in drinks and chutneys. A tamarind seed pod is
shown in Figure 4. In tamarind seeds large amounts of on
xyloglucan dramatically change the physical properties of the
polymer are found in relative high purity. Currently kernel powder
(TKP) from Tamarindus indica is available in different qualities in
bulk quantities from India.
In TKP about 60% of the dry weight is on xyloglucan
dramatically change the physical properties of the polymer, TKP is
also available in a de-oiled variant (DTKP) (Shankaracharya,
1998). DTKP is slightly easier to dissolve in water and stays
odorless even during longer storage periods. Tamarind on
xyloglucan dramatically change the physical properties of the
Figure 4. Left: The seed pod of Tamarindus indica the woody outer shell is opened and the dark brown pulp is visible. Right: Close-up, the seeds are embedded in the acidic pulp and have a brown skin. (Photos taken by Martin Baumann)
Xyloglucan is a cell wall polysaccharide
13
polymer is not fucosylated (York et al., 1993). A number of
Indian companies specialize in supplying TKP; extracted on
xyloglucan dramatically change the physical properties of the
polymer can be bought in different chain length from Japanese
companies.
3.2. Xyloglucan solution properties and applications Xyloglucan has remarkable solution properties, recently
reviewed by (Yamatoya and Shirakawa, 2003). Xyloglucan
solutions with a concentration up to 5 g/L are easy to handle. Its
aqueous solutions exhibit Newtonian fluidity (Gidley et al., 1991).
The solution characteristics of xyloglucans extracted from different
plant species, such as tamarind seed (Gidley et al., 1991), apple
pomace and the extra cellular medium of suspension cultured
Nicotiana plumbaginifolia cells (Sims et al., 1998), have been
compared. The length of glucose backbone of the xyloglucan
oligosaccharide is the major factor influencing the solution
properties of XGs. With the growing chain length the viscosity
increases. The chain length can be easily manipulated by endo-
xyloglucanases (Vincken et al., 1996a; Vincken et al., 1997a;
Pauly et al., 1999c; Irwin et al., 2003; Yaoi et al., 2004; Cho et
al., 2006; Martinez-Fleites et al., 2006; Gloster et al., 2007) like
CtXGH74A (Paper 1) or NXG1 (Paper 2, Edwards et al., 1985;
Paper 2, Edwards et al., 1988). Even though xyloglucan is highly
water soluble, in the presence of saccharose, alcohol or gellan
xyloglucan forms gels (Yamanaka et al., 2000; Marathe et al.,
2002; Salazar-Montoya et al., 2002; Nitta et al., 2003; Ikeda et
al., 2004; Yuguchi et al., 2004). The enzymatic removal of
galactose from the xyloglucan side chains has a similar effect
(Shirakawa et al., 1998).
Martin J. Baumann
14
Polysaccharide gels can be used as carriers for
pharmaceutical delivery, providing a constant dosage of the drug
(Miyazaki et al., 1998; Miyazaki et al., 2003; Yamatoya and
Shirakawa, 2003). The high solubility of xyloglucan in water and
its rheological properties makes it a useful food additive and it is
used as a thickener, stabilizer and gelling agent, primarily in Asia.
(Miyazaki et al., 1998; Yamatoya and Shirakawa, 2003).
xyloglucan strongly retains water, and the addition of hydrolyzed
xyloglucan dramatically improves the hardness and reduces water
release of freeze-thawed carrageenan gel (Shankaracharya, 1998;
Yamatoya and Shirakawa, 2003).
A boiled DTKP solution can be used for textile sizing of jute
or cotton (Rao and Srivastava, 1973; Shankaracharya, 1998). A
recent application is enzymatic stone washing. The cotton yarn is
pre-coated with xyloglucan before dying, and removal of the
xyloglucan by endo-glucanase treatment gives the fabric a used
look by partial dye removal (Kalum, 1998).
It has been described that xyloglucan can be used in paper
making, replacing starch or galactomanan (Shankaracharya,
1998). Lately this idea has been further investigated and
xyloglucan has been tested as a wet end additive (Christiernin et
al., 2003; Lima et al., 2003). The addition of xyloglucan to the
pulp reduces the friction of the fibers and may thereby improve
sheet formation (Stiernstedt et al., 2006).
Xyloglucan can be digested with specific xyloglucanases to
xyloglucan-oligosaccharides (XGOs). XGOs can be used for cell
wall analysis (Sims et al., 1996; Pauly et al., 1999a; Hoffman et
al., 2005a). Fluorophoric XGOs can be can be used to detected
Xyloglucan active enzymes (e. g. XETs) (Bourquin et al., 2002), or
for kinetic studies (Fauré et al., 2006). XGOs can also be used as
Xyloglucan is a cell wall polysaccharide
15
hydrophilic head group for surfactant synthesis (Greffe et al.,
2005).
3.3. Interaction of xyloglucan and cellulose There has been a long interest in the interaction of
xyloglucan and cellulose (a selection of experimental studies is
presented in Table 3), since the 1980s when Hayashi and co-
workers started to reconstitute extracted xyloglucan from pea
onto pea cellulose (Hayashi and Maclachlan, 1984; Hayashi et al.,
1987). Later, xyloglucan of various degrees of polymerization
from T. indicia were used to anneal to amorphous cellulose
(Hayashi and Takeda, 1994; Hayashi et al., 1994b). The binding
conditions were with 120°C rather harsh and the used xyloglucan
oligosaccharides were smaller than the oligosaccharides used in
subsequent publications. Under physiological conditions like pH
5.8 and 40°C, the minimum chain length is four to five XGOs
(structure of XGO Figure 3), i. e. a xyloglucan oligosaccharide with
a Glc16 to Glc20 backbone length (Vincken et al., 1995; Hanus and
Mazeau, 2006). Binding of xyloglucan to cellulose occurs in a
wide range of pH (2 to 8) and temperature (5-60°C) (de Lima and
Buckeridge, 2001). Xyloglucan binds to various types of cellulose
surfaces, such as bacterial cellulose, plant cellulose and
regenerated cellulose and wood pulps (references in Table 3).
Xyloglucan does not bind to cellulose at conditions of high pH
typically used for extraction from plant material.
The exact interaction of xyloglucan with cellulose is not
understood; it is believed that xyloglucan binds through non-
covalent interactions to cellulose. In single molecule force
spectroscopy studies (AFM), the bonding energy between
xyloglucan and pure crystalline cellulose has been studied. In
combination with modeling the binding energy was estimated to
Martin J. Baumann
16
be approximately equivalent to one H-bond per glucose unit
(Morris et al., 2004).
Table 3. Selected binding studies of xyloglucan to cellulosic materials Xyloglucan source
cellulose source and binding conditions
Reference
Tamarindus indica bleached chemical and mechanical wood pulps
(Zhou et al., 2006a)
Tamarindus indica
Avicel
(Zykwinska et al., 2005)
Rubus fruticosus suspension cultures at different time points of growth curve
Primary cell wall, cotton, flax, bacterial cellulose, tuniciae, rhizoclonium, quantification of unbound XG
(Chambat et al.,
2005)
Tamarindus indica
Filter paper, binding at 20°C, in pure water, quantification of FITC labeled XG on the paper surface, or of remaining XG in solution
(Brumer et al., 2004)
Phaseolus vilgaris L (common bean) C. langsdorfii, Tamarindus indica and H. courbaril
Microcrystalline cellulose Binding at 30°C, pH 6.0, 15 Min., quantification of remaining XG in solution
(Lima et al., 2004)
Phaseolus vilgaris L (common bean) C. langsdorfii, Tropaeolum majus, Tamarindus indica and H. courbaril
Microcrystalline cellulose, Binding at 5-60°C, pH 2-8, 15 Min., quantification of remaining XG in solution
(de Lima and Buckeridge, 2001)
Tamarindus indica oligosaccharides (1-11 repeating units and high MW)
Avicel and cotton linters Annealing at 40°C pH 3 and 5.8, 6h, quantification of remaining XG in solution
(Vincken et al., 1995)
Pea (pisum sativum L. Var. Alaska) Pea cellulose, XG extracted and reconstituted, immuno-gold detection
(Baba et al., 1994)
Tamarindus indica and Pisum sativum high MW down to 1000 KDa
Amorphous cellulose (PASC) annealing at 30°C –120°C, carbohydrate quantification after binding and re-extraction of XG from formed complex.
(Hayashi et al., 1994a)
Tamarindus indica, oligosaccharides (DP8-12 and 3000-64) 5 pisum sativum (DP 150) 4 and cello oligosaccharides (DP5-6) 4
Amorphous cellulose, microcrystalline cellulose, annealing at 120°C 10 Min., pH 5.5, 3H labeled scintillation counting of bound radioactivity. Binding isotherms of XGO and cello oligosaccharides
(Hayashi et al., 1994a)
Tamarindus indica, oligosaccharides (DP3-12) 4 and cellooligosaccharides (DP2-6) 1
Amorphous cellulose, microcrystalline cellulose, filter paper, annealing at 120°C 10 Min., pH 5.5, 3H labeled scintillation counting of bound radioactivity. Binding isotherms of XGO and cello oligosaccharides
(Hayashi et al., 1994b)
Pine (pinus pinaster) from seedlings Commercial cellulose (CF-11 or CF-41 Whatman), pine cellulose, 40°C, 15 h, pH 5.5, quantification of remaining XG in solution, quantification of released xyloglucan, Visualization with fluorescein labeled fucose binding lectin, CMC viscometry
(Acebes et al., 1993)
Pea (pisum sativum) Pea cellulose, Acetobacter xylinum cellulose, Valonia ventricosa cellulose, Binding at pH 5, 40°C, 6h, radioactive detection (125I) or fluorescent labeled XG, competition experiments with other hemicelluloses
(Hayashi et al., 1987)
Pea (pisum sativum L. Var. Alaska), Tamarindus indica and cellopentaose
Pea cellulose (cell wall ghosts) Binding visualization with fluorescein labeled fucose binding lectin (U. europeus), radioactive label (3H) and iodine staining of XG-cellulose complex – and reconstitution in vitro
(Hayashi and Maclachlan, 1984)
5 Number of 1,4-β-glycosyl units
Xyloglucan is a cell wall polysaccharide
17
In silico studies in suggest that xyloglucan can bind to all
sides of crystalline cellulose (Hanus and Mazeau, 2006), binding
studies with modified xyloglucans showed, that binding is
independent from galactose or fucose side chain substitutions
(Chambat et al., 2005). Most likely the xyloglucan chain adopts a
stretched linear confirmation, and the side chains extend over the
cellulose surface (Hanus and Mazeau, 2006).
Martin J. Baumann
18
4. Carbohydrate active enzymes in the biosynthesis and degradation of xyloglucan
A number of glycosyl transferases (GT) involved in
xyloglucan biosynthesis have been identified; they are discussed
in chapter 4.2. Xyloglucan-active degrading enzymes are found in
several GH families: GH5 (Gloster et al., 2007), GH12 (Pauly et
al., 1999b; Gloster et al., 2007), GH16 (Edwards et al., 1985;
Johansson et al., 2004; Kallas et al., 2005), GH 26 (Cho et al.,
2006), GH44 (Schnorr et al., 2001) and GH74 (Irwin et al., 2003;
Yaoi et al., 2004; Martinez-Fleites et al., 2006). The wide
distribution of xyloglucan-active enzymes suggests that this
specificity has been developed repeatedly during evolution. These
carbohydrate active enzymes will be discussed in subsequent
sections, following a brief introduction to enzyme nomenclature.
4.1. Enzyme classification Enzymes can be classified by a number of criteria; currently
two systems are used in parallel for carbohydrate active enzymes:
the EC-number classification and CAZy.
4.1.1. Enzyme nomenclature according to NC-IUBMB6
The International Union of Biochemistry and Molecular
Biology (IUBMB) was established in 1956 and has since then
provided a systematic classification of enzymes (Webb, 1992). A
new enzyme will be assigned to a class only by its main activity.
Furthermore, the reaction catalyzed, the bond cleaved and the
substrate utilized are encoded in the EC number. The enzyme
6 NC-IUBMB: Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, http://www.chem.qmul.ac.uk/iubmb/enzyme/
Xyloglucan-active enzymes
19
classification system by the NC-IUBMB also includes rules for
enzyme nomenclature. One or several accepted names are
assigned to each EC number. The six main classes are: EC 1
Oxidoreductases, EC 2 Transferases, EC 3 Hydrolases, EC 4
Lyases, EC 5 Isomerases and EC 6 Ligases, the following numbers
stand for subclasses, the EC numbers for xyloglucan endo-
hydrolases and xyloglucan endotransferases are explained in Table
4, EC numbers of xyloglucan-active enzymes are listed in Table 5.
Table 4. Explanation of EC subclasses for xyloglucan endohydrolases and xyloglucan endotransferses EC number Name of subclass
3 Hydrolases
3.2 Glycosylases
3.2.1 Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds
3.2.1.151 Xyloglucan-specific endo-β-1,4-glucanase
2 Transferase
2.4 Glycosyl-transferase
2.4.1 Hexosyltransferases
2.4.1.207 Xyloglucan:xyloglucosyl transferase
The advantage of the EC system is its simplicity; the only
initial information needed is the enzyme activity. The system has
therefore been extensively applied and is well established. The
nomenclature system is however somewhat limited when applied
to enzymes with a broad substrate range, or enzymes with several
substrates. Furthermore the EC numbers reflect evolutionary
information or mechanistic information only to a very limited
extent. Finally no information about required cofactors or enzyme
structures is included in the EC number.
Martin J. Baumann
20
Table 5. Selected xyloglucan-active enzymes and their EC-numbers. EC number activity Selected reference
EC 3.2.1.151 xyloglucan-specific endo-β-1,4-glucanase (Pauly et al., 1999c)
EC 3.2.1.155 xyloglucan-specific exo-β-1,4-glucanase (Grishutin et al., 2004)
EC 3.2.1.150 oligoxyloglucan reducing-end-specific cellobiohydrolase
(Yaoi et al., 2004)
EC 2.4.1.207 Xyloglucan endotransferase (Fry et al., 1992)
EC 2.4.1.168 xyloglucan 4-glucosyltransferase (Hayashi et al., 1980; Hayashi and Matsuda, 1981)
EC 2.4.2.39 xyloglucan 6-xylosyltransferase (Faik et al., 2002)
2.4.1.- xyloglucan 2-galactose transferase (Madson et al., 2003; Li et al., 2004)
Xyloglucan-active enzymes
21
4.1.2. Carbohydrate-Active enzymes7
The CAZy classification applies to carbohydrate active
enzymes only. Enzymes are divided into four main classes,
according to the type of reaction catalyzed: GH glycosyl
hydrolases (GH, Henrissat and Davies, 1997), glycosyl
transferases (GT, Coutinho et al., 2003), polysaccharide lyases
(PL) and carbohydrate esterases (CE, Coutinho and Henrissat,
1999). A special class are the non-catalytic carbohydrate binding
modules (CBM, Boraston et al., 2004). Within each main class,
enzymes are grouped into families according to their sequence
similarity (Henrissat and Davies, 1997). Because of sequence
similarities, certain features are shared within one family, the
overall 3-D structure, and also the catalytic mechanism and
machinery. However the substrate specificity within one family
can be rather diverse. Moreover, some families are clustered into
clans, each clan representing a general fold pattern.
4.2. Xyloglucan biosynthesis in the plant cell Like all hemicelluloses, xyloglucan is synthesized in the Golgi
apparatus in the cell lumen, transported in vesicles and finally
secreted as a polymer in the cell wall (Reiter, 2002; Scheible and
Pauly, 2004; Lerouxel et al., 2006). According to the current
model of xyloglucan biosynthesis a β(1-4)-glucan chain is
produced by a cellulose synthase-like protein (CSL) or a protein
complex located in the outer membrane of the Golgi. This protein
has not yet been identified, and it is unknown if the nucleotide
sugars are originating from the cytosol or Golgi lumen (Figure 5).
The β(1-4)-glucan polymer is secreted into the lumen and
decorated with xylose by xylosyl transferases, which are
7 http://afmb.cnrs-mrs.fr/CAZY/ or www.cazy.org
Martin J. Baumann
22
responsible for the highly regular substitution pattern. It is not
yet known if the decoration with xylose is performed upon
secretion, or if the xylosyl transferases act in a complex with the
CSL protein. Two of the xylosyl transferases from arabidopsis
have been identified (Faik et al., 2002). Both belong to GT34 (EC
2.4.2.39) and in vitro activity has been demonstrated after
heterologous expression in insect cells (Cavalier and Keegstra,
2006). Both proteins (AtXT1 and AtXT2) have similar specificity,
they add xylose to cellohexanose to the forth and third glucose
residue form the reducing end.
NDP-NDP
NDP-
NDP
Golgi
membrane
Golgi lumen
Cytosol
NDP-NDP
CSL CSL
GT
Figure 5. Two models of hemicellulose synthesis in the Golgi. Left: A cellulose synthase like protein (CSL) is a transmembrane protein in the Golgi membrane, it polymerizes the glucan chain from nucleotide sugars originating from the Golgi lumen. Right: A cellulose synthase like protein (CSL) polymerizes a glucan chain from nucleotide sugars originating from the cytosol, glycosyl transferase (GTs) are in close proximity and add side chains. Figure adapted after (Lerouxel et al., 2006)
Furthermore, a galactosyl transferases (MUR3, belongs to GT47)
add β(1→2)-Gal to xyloglucan (Madson et al., 2003; Li et al.,
2004). A fucosyl transferase, called AtFUT1 (Perrin et al., 1999)
has been identified as a GT47 protein too. The side chains of
xyloglucan may vary between tissues (2.2), this can be explained
by tissue specific expression or activity of the previously described
GTs (Hayashi, 1989).
Xyloglucan-active enzymes
23
4.3. GH hydrolases Currently over 100 GH families are known. The enzymes so
far studied have 14 different general folds and hydrolyze
approximately 150 different substrates (as estimated from known
EC numbers). Even though the substrates seem to be extremely
diverse, all GH enzymes split a glycosidic bond. Presumably, most
GH families utilize either the retaining or the inverting mechanism.
The reaction mechanism of about 85 GH families is known: 36
utilize the inverting mechanism and 46 utilize the retaining
mechanism. In few special cases kinetically-controlled
transglycosylase activity of GH enzymes using the retaining
mechanism has been observed (4.6 page 29).
XGOs (Figure 3) can be degraded to monosaccharides by GH
enzymes. In principle for each GT exists at least one GH. Several
β-galactosidases are known (Edwards et al., 1988; de Alcântara et
al., 2006). Also, xylosidases have been studied (Fanutti et al.,
1991; Trincone et al., 2001). The glucose backbone can be
broken down by endoglucanases in the same way as cellulose
(Schülein, 2000).
4.4. Inverting xyloglucan-hydrolases Inverting xyloglucan hydrolases (EC 3.2.1.151) are found in
GH74 (Martinez-Fleites et al., 2006) and possibly in GH44
(Schnorr et al., 2001). In both cases the proteins originate from
bacteria.
Martin J. Baumann
24
4.4.1. Inverting mechanism
The inverting mechanism is used by, for example, GH74
(Paper 4). The inverting mechanism is a direct displacement
mechanism, resulting in the opposite (inverted) configuration on
the anomeric carbon (Figure 6). The mechanism passes through
one transition state, and no glycosyl-enzyme intermediate is
formed. In the beginning of the reaction a general base activates
a water molecule by abstracting a proton. The resulting partial
negative charge enables the oxygen to perform a nucleophilic
attack on the anomeric carbon. At the same time, the leaving
group is activated by general acid catalysis through donation of a
proton to the departing oxygen. An oxocarbenium ion-like
transition state is formed (Vocadlo et al., 2001). In this transition
state electron density is distributed over no less than 8 atoms,
and three bonds are broken and formed almost simultaneously.
Enzymes employing this reaction mechanism generally exhibit a
distance of approximately 11 Å between the catalytic residues.
This is the distance required to accommodate the substrate and a
water molecule in the transition state (Figure 6). (Withers, 2001)
OHOHO
OHO
OH
R
OO
HO O
HO H
OHOHO
OH
O
OH
OO
O O
HOH
OHOHO
OHOH
OH
OHO
O OH
R
O
H
R
Figure 6. The inverting transglycosylation mechanism, adapted from Withers (2001), the distance between the general base and the general acid is typically 11 Å.
Xyloglucan-active enzymes
25
4.4.2. Inverting xyloglucan hydrolases in the partial enzymatic degradation of plant cells wall by bacteria
Plant cell walls are a natural composite material, and several
different enzyme activities are required for hydrolysis. Aerobic
bacterial and fungi secrete individual enzymes. Anaerobic
cellulolytic bacteria like Clostridium thermocellum produce a large
extra cellular cellulase system called the cellulosome (Bayer et al.,
2004). The cellulosome is an extremely intricate protein complex
consisting of over 20 different catalytic subunits ranging in size
from about 40 to 180 kDa, with a total molecular weight of a
million kDa. Besides cellulolytic enzymes, the presence of
hemicellulolytic enzymes has also been shown: for example, a
mannanase, a β-xylosidase (Desvaux, 2005 and references
therein) and an inverting xyloglucanase CtXGH74A (Paper 4).
The key feature of the cellulosome is the nonhydrolytic
"scaffolding" subunit that integrates the various catalytic subunits
into the complex through interactions between its repetitive
"cohesin" domains and complementary "dockerin" domains on the
catalytic subunits. It is now known that many anaerobic different
cellulolytic bacteria and fungi produce extra cellular multi-enzyme
complexes similar to the cellulosome. The C. thermocellum
cellulosome is the best-characterized cellulase complex, and thus
serves as a paradigm.
Martin J. Baumann
26
4.5. Retaining xyloglucan hydrolases All enzymes in the glycosyl hydrolase family 16 are believed
to utilize the double displacement mechanism for catalysis. Two
sequential nuclephilic substitutions on the anomeric carbon result
in an overall retention of the anomeric configuration (Sinnott,
1990).
4.5.1. Retaining mechanism of GH16 hydrolases
The double displacement mechanism passes through two
transition states. In the first step, a deprotonated carboxylic acid
acts as a nucleophile attacking the anomeric C1 to form a covalent
glycosyl-enzyme intermediate. At the same time, another
carboxylic acid side chain residue acts as a general acid, and
protonates the leaving group. In the second step of the reaction
mechanism the very same amino acid residue acts as a general
base to deprotonate water. The activated water attacks the
covalent glycosyl-enzyme intermediate and breaks it down.
(Vocadlo et al., 2001).
OHOHO
OHO
OH
R
OO
HO OOHO
HOOH
OH
OO
O OOHO
HOOH
OH
OH
OO
HO O
HO H
Figure 7. The retaining hydrolysis mechanism, adapted from Withers (2001), the distance between nucleophile and acid/base is typically 5.5 Å.
Xyloglucan-active enzymes
27
4.5.2. Biological role of GH16 hydrolases in plants
It has been shown that the molecular weight of xyloglucan
decreases during fruit ripening, possibly caused by cell wall active
XG-hydrolases (Schröder et al., 1998; Rose et al., 2002). The
only xyloglucan-hydrolyzing enzyme from GH16 characterized in
detail is NXG1. Originally isolated from nasturtium cotyledons by
Edwards et al. (1985; 1986), the hydrolytic activity of the enzyme
on xyloglucan was discovered. It was further revealed by Fanutti
et al. (1993) that NXG1 had xyloglucan endo-transglycosylating
activity. The DNA sequence was subsequently cloned by De Silva
et al (1993). A detailed study analyzing the binding site
requirements followed by Fanutti et al. (1996).
A number of close homologs of NXG1 have been analyzed by
expression profiling. Surprisingly they have been shown to be
expressed during different stages of the plant development. This
indicates a versatile role for putative xyloglucan hydrolases during
plant development. AtXTH31 and AtXTH32 from Arabidopsis are
closely related to NXG1. AtXTH31 (82% similarity, 67% identity
to the protein sequence of NXG1) is expressed in germinating
seeds and during the later plant development in roots and flower
buds (Aubert and Herzog, 1996). Also, Yokoyama et al. (2001)
found AtXTH31 expression in roots and stem, and AtXTH32 (82%
similarity, 69% identity to NXG1) in the stem. The
β-glucuronidase (GUS) from E. coli has been used as a reporter
protein in gene fusion studies to investigate tissue specific gene
expression (Jefferson et al., 1987b; Jefferson et al., 1987a). In
GUS-fusion studies high expression levels of AtXTH31 have been
found in the root elongation zone (Becnel et al., 2006).
Tomato (Solanum lycopersicum) 8 SlXTH6 is also closely
related to NXG1 (82% similarity, 71% identity), EST clones of
8 Also called Lycopersicon esculentum
Martin J. Baumann
28
SlXTH6 have been found in libraries from the developing shoot,
germinating seeds, and roots (Saladie et al., 2006). In grape
berries (Vitis labrusca x Vitis vinifera), VXET1 (85% similarity,
73% identity to NXG1) expression is induced during berry
softening (vérasion) (Ishimaru and Kobayashi, 2002).
Several gene sequences in rice (Oryza sativa) have high
similarity to NXG1. Among them is OsXTH19 9 (75% similarity,
61% identity to NXG1) and OsXTH21 (76% similarity, 62%
identity to NXG1), these genes are at least expressed in leaves
(Yokoyama et al., 2004).
4.6. Retaining GH transglycosylases Among the hydrolytic GH16 enzymes are surprisingly also
strict transglycosylases found. All GH16 transglycosylases
catalyze endo-transglycosylation of xyloglucan (EC. 2.4.1.207).
According to CAZy, currently more than 28 proteins 10 are
designated as xyloglucan endo-transglycosylases, all from plants.
The assigned EC number is used as selection for enzyme activity
in the following chapter.
4.6.1. Mechanism of retaining xyloglucan endotransferases
The transglycosylating reaction mechanism is essentially the
same as for retaining hydrolases, based on a double displacement
mechanism passing through two transition states. In the first
step, a deprotonated carboxylic acid acts as a nucleophile
attacking the anomeric center to form a covalent glycosyl-enzyme
intermediate. At the same time, another carboxylic acid side
9 OsXTH20 is also closely related to TmNXG1, but currently no expression data are published. 10 Information taken from the CAZy web site in February 2007.
Xyloglucan-active enzymes
29
chain acts as a general acid and protonates the leaving group. In
the second step of the reaction mechanism, the very same amino
acid residue acts as a general base to deprotonate the 4´hydoxy
group on the non-reducing end of a xyloglucan glycosyl acceptor.
Surprisingly water is not utilized in this step. The nucleophilic
attack on the anomeric carbon breaks the enzyme-glycosyl
intermediate and a new glycosidic bond is formed (Vocadlo et al.,
2001).
OHOHO
OHO
OH
R
OO
HO OOHO
HOOH
OH
OO
O OOHO
HOOH
O
OH
R'
OO
HO O
HO R'
Figure 8. The retaining transglycosylation mechanism, adapted from Withers (2001), R’= represents a transglycosylation acceptor. The distance between nucleophile and acid/base is typically 5.5 Å.
4.6.2. Retaining transglycosylases among GH enzymes
Several enzymes of the GH families are known to show
transglycosylase activity under artificial conditions like low water
content or high substrate concentrations (Crout and Vic, 1998;
York and Hawkins, 2000). Strict transglycosylase activity with no
detectable hydrolysis activity under physiological conditions is a
rare but outstanding phenomenon for GH enzymes. Only very few
of all studied GH enzymes are actually assigned to EC class of
transglycosylases, including XETs (2.4.1.207).
From about 150 activities 11 only 28 glycosyl-transferring
activities are found (that is EC numbers beginning with 2.4.1.-).
Even though the assignment of EC number may not be
unambiguous in all details, it can be used as a guideline
11 according to assigned EC numbers on the CAZy website in 02/2006
Martin J. Baumann
30
considering the large amount of data. Transglycosylating activity
is found in 15 GH families (13 retaining and 2 inverting, Table 6),
some families are most likely exclusively transglycosylating (e.g.
GH 70).
The inverting transglycosylases (in GH 65 and GH 95) are unique,
instead of a water molecule which is used by inverting hydrolases,
inorganic phosphate is required (4.4.1 page 24) (Stoffer et al.,
1995; Egloff et al., 2001; Hidaka et al., 2006).
Table 6. Examples of transglycosylating enzymes currently known from the retaining GH families. Examples were taken from the CAZY web site12 and selected by EC number (2.4.1.-). EC number GH
family Reaction
mechanism Enzyme activity Selected References
2.4.1.19 2.4.1.18 2.4.1.25 2.4.1.4 2.4.1.7
13 retaining cyclomaltodextrin glucanotransferase branching enzyme 4-α-glucanotransferase Amylosucrase sucrose phosphorylase
(Lawson et al., 1994) (Abad et al., 2002) (Roujeinikova et al., 2002) (Mirza et al., 2001) (Sprogoe et al., 2004)
2.4.1.207 16 retaining xyloglucan endotransferase
(Johansson et al., 2004)
2.4.1.- 17 retaining β-1,3-glucan transglycosidases
(Mouyna et al., 1998)
2.4.1.- 31 retaining Isomaltosyltransferase
(Aga et al., 2002)
2.4.1.99 2.4.1.100
32 retaining sucrose:sucrose 1-fructosyl transferase fructan fructan 1-fructosyltransferase
(Vijn et al., 1998) (van der Meer et al., 1998)
2.4.1.- 33 retaining trans-sialidase
(Amaya et al., 2004)
2.4.1.67 2.4.1.82
36 retaining stachyose synthase raffinose synthase
(Peterbauer et al., 2002)
2.4.1.25 57 retaining 4-α-glucanotransferase
(Laderman et al., 1993)
2.4.1.- 66 retaining cycloisomaltooligosaccharide glucano-transferase
(Oguma et al., 1994)
2.4.1.10 2.4.1.9
6813 retaining levansucrase inulosucrase
(Meng and Futterer, 2003) (van Hijum et al., 2002)
2.4.1.5 2.4.1.140
7014 retaining Dextransucrase Alternansucrase
(Kang et al., 2005) (Arguello-Morales et al., 2000)
2.4.1.- 7215 retaining β-1,3-glucanosyltransglycosylase
(Mouyna et al., 2000)
2.4.1.25 7716 retaining amylomaltase or 4-α-glucano-transferase
(Przylas et al., 2000)
12 http://afmb.cnrs-mrs.fr/CAZY/ or www.cazy.org 13 Almost all currently characterized members (36 of 40) of GH68 are trans-glycosylating. 14 All currently characterized members (36 of total 56) of GH70 are trans-glycosylating. 15 All currently characterized members (4 of total about 83) of GH72 are trans-glycosylating. 16 All currently characterized members (16 of total more than 250) of GH77 are transglycosylating.
Xyloglucan-active enzymes
31
4.6.3. Biological roles of GH16 enzymes in cell expansion
XET activity has been detected in vivo in a wide variety of
plants and tissues (Vissenberg et al., 2000; Bourquin et al., 2002;
Vissenberg et al., 2003; Fry, 2004; Van Sandt et al., 2006). XETs
belong to the XTH gene subfamily, which includes hydrolytic XG-
hydrolases and transglycosylating XETs (Rose et al., 2002). XTH
genes are found in large families: for example 33 sequences in
Arabidopsis (Yokoyama and Nishitani, 2001), 29 sequences in rice
(Yokoyama et al., 2004), 36 gene models in the Populus
trichocarpa genome draft (Geisler-Lee et al., 2006) and 25
sequences in tomato EST libraries (Saladie et al., 2006). The XTH
gene family members are expressed tissue specifically during
plant development, suggesting that genes are regulated
individually (Becnel et al., 2006; Saladie et al., 2006).
Some XTH gene products might be involved in cell wall
restructuring, others in incorporation of newly synthesized
xyloglucan into the cell wall, and possibly yet others in hydrolyzing
xyloglucan. XETs have the potential to modify the cellulose-
xyloglucan network, thus XETs are most likely involved in the
reconstruction of cell walls during cell expansion (Carpita and
Gibeaut, 1993). A transient cutting and religation, as performed
by XETs, would be feasible for maintaining the strength of the
primary cell wall. During cell expansion, the surface area of the
cell wall increases dramatically. In spite of this, the thickness of
the cell wall is kept constant, which means that new cell wall
material must be incorporated continuously during cell expansion.
It has been shown in vivo that newly synthesized xyloglucan is
added to the cell wall xyloglucan pool (Thompson and Fry, 2001).
Most likely, XETs are involved in this process.
Recently it was shown that XET activity could be detected
from the primary cell wall to the early development of the
Martin J. Baumann
32
secondary cell wall. In situ assays of XET in hybrid aspen have
shown that XET activity can be found in xylem and phloem fibers
during deposition of the secondary cell wall. Immunolocalizion
with antibodies raised against PttXET16-34 (renamed to PttXET16-
34 after Geisler-Lee et al., 2006) from hybrid aspen confirmed the
presence of XETs in these tissues at this developmental stage
(Bourquin et al., 2002). However, since hybrid aspen has
approximately 41 members of the XTH gene family it is currently
unclear which XET isozymes were visualized.
Aims of the present investigation
33
5. Aims of the present investigation
The general objective was to perform detailed structure-function
studies of xyloglucan active enzymes, with special focus on
xyloglucan active enzymes from GH16.
The specific goals were:
• Preparation of xyloglucan oligosaccharides and the
synthesis of aryl-xyloglucan oligosaccharides as kinetic
probes, and/or as ligands for protein crystallography.
• Assay development for quantification of hydrolytic and
transglycosylation activity of xyloglucan active enzymes.
• Comparative structure-function studies of xyloglucan
endotransferases and xyloglucanases.
• Application of XET for biomimetic fiber surface
modification, including preparation of xyloglucan
oligosaccharides with versatile functional groups for fiber
modification.
Martin J. Baumann
34
6. Results and discussion This thesis comprises characterization of xyloglucan-active
enzymes (Papers 1-4). Three protein structures (Table 7) from
two different GH families are presented (Papers 1-3). Two of
these structures are from plant enzymes (Papers 2 and 3).
Furthermore, the protein studied in Paper 3 is applied in a
biomimetic method for fiber modification (Paper 5).
Table 7. Structures presented in this work
Enzyme GH
family
source activity PDB id
apo-structure
PDB id
ligand-structure
CtXGH74A GH74 Clostridium thermocellum Xyloglucanase
(3.2.1.151)
2CN2 2CN3
NXG1 GH16 Tropaeolum majus Xyloglucanase /XET
(2.4.1.207/ 3.2.1.151)
In preparation -
PttXET16-34 GH16 Populus tremula x
tremuloides
XET
(2.4.1.207)
1UN1 1UMZ
6.1. CtXGH74A structure and properties The protein CtXGH74A is part of the Clostridium
thermocellum cellulosome. The C-terminal dockerin domain was
removed and the catalytic domain expressed heterologously in E.
coli (Paper 1, Figure 2 A and B). CtXGH74A is a specific XG-
hydrolase with some activity on carboxymethyl cellulose, lichenan
and hydroxyethyl cellulose (Paper 1, table 1). The xyloglucan
chain is cleaved in an endo fashion at the anomeric carbon of the
unbranched G unit (EC 3.2.1.151, Paper 1, Figure 3; structure of
XGO Figure 3, page 9). Product analysis by HPAEC-with PAD
detection and ESI-TOF MS does not show any preference for any
Results and Discussion
35
of the four different XGOs or any occurrence of oligosaccharides
smaller than Glc4 based XGOs (Paper 4, Figure 4).
Structure determination by X-ray crystallography shows that
the CtXGH74A has two domains, each domain composed of a
seven fold β-propeller. The two seven fold β-propellers are
positioned at an angle of approximately 90º to each other (Paper
4, Figure 5). The substrate binding site is an open cleft situated
at the intersection of the two domains. The cleft runs along the
whole intersection and is formed by loops connecting the β-
propeller blades from both domains.
Catalysis by GH74 enzymes follows the inverting mechanism
(4.4.1, page 24). The inferred catalytic residues in CtXGH74A are
aspartate 70 and aspartate 480 (D70 and D480). The distance
between the residues is approximately 10 Å, as expected for an
inverting enzyme. The catalytic residues are positioned in the
middle of the active site cleft, at opposite sides deep in the cavity.
The structure was solved for the wild type apo-enzyme (PDB id
2CN2) and a nucleophile mutant (D70A) in complex with two XGO
molecules bound in the active site (PDB id 2CN3). In the complex
structure, several previously unordered loops adopted an ordered
confirmation and contributed to substrate binding. Electron
density for 17 sugars was observed in the complex (Paper 4
Figure 6A). One molecule of XLLG and one molecule of XXLG
(short hand notation of XGOs; 2.2, Page 9) was bound in the
active site. The XGOs are bound in an extended confirmation with
an almost linear glucose backbone and the xylose residues are
pointing outwards to opposing sides, the ring planes of the xylose
side chains are almost perpendicular to the ring plane of the
glucoses. The glucose units in the positive and the negative
subsites are at an angle of about 110° to each other when
measured at the glucose backbones of the XGOs. In total, four
Martin J. Baumann
36
positive subsites and four negative subsites can be assigned
(Paper 1, Figure 6B), and the protein interacts through many
hydrogen-bonds with the ligand (Paper 4, Figure 6B and 7). The
structure shows that several conserved residues in GH74 are
involved in the xyloglucan binding (Paper 1, Figure 8).
Currently one additional protein structure of GH74 has been
solved, the oligoxyloglucan reducing end-specific cellobiohydrolase
from Geotrichum sp. M128 (OXG-RCBH, PDB id 1SQJ, 36%
sequence identity to CtXGH74A, 50% similarity). OXG-RCBH
cleaves xyloglucan in an exo-fashion and an oligosaccharide with a
backbone length of two glucose units is released (EC 3.2.1.150).
Comparing of OXG-RCBH to CtXGH74A, the active site cleft of
OXG-RCBH is blocked by a loop in the +3 subsite (Paper 1 Figure
9B), that might be the major determinant for the exo-mode of
action of OXG-RCBH.
6.2. NXG1 structure and properties It has been reported that NXG1 is a protein of the XTH gene
family with hydrolytic activity (Rose et al., 2002). Originally the
protein was isolated from hypocotyls of nasturtium seeds
(Edwards et al., 1985). In this study we cloned and expressed
two single amino acid polymorphs, NXG1 and NXG2, in Pichia
pastoris. For protein purification an affinity chromatography
method was developed, with aminated XGO (Paper 5) immobilized
on a NHS-preactivated column. Pure NXG1 protein shows activity
on xyloglucan, and exhibited no detectable activity on konjac
glucomannan, Icelandic moss lichenan, barley β-glucan, Avicel
and birchwood xylan. A trace amount of activity was observed
with hydroxyethyl cellulose, as was previously shown for an
extracted and purified enzyme preparation (Edwards et al., 1985).
xyloglucan is cleaved hydrolytically in an endo fashion (Paper 3,
Results and Discussion
37
Figure 1, A). The depolymerisation of xyloglucan by NXG1 is
inhibited in the presence of XGO (Paper 2, Figure 1B). In
sequence alignments, two Loop regions close to the active site
(Paper 2, supplemental figure protein sequence alignment) were
identified as being divergent in the hydrolytic NXG1 and the
transglycosylase PttXET16-34 (Paper 2, Paper 3). Mutants for
both Loops (Loop 1 and Loop 2) were designed, but only the Loop
2 mutant (NXG1-ΔYNIIG) was obtained as expressed protein.
NXG1-ΔYNIIG shows reduced hydrolytic activity on xyloglucan
(Paper 2, Figure 2C) and the depolymerization of xyloglucan was
sped up in the presence of XGO (Paper 2, Figure 2D). The
catalytic behavior was further analyzed with a Glc8-based XGO as
the substrate. For initial rate kinetics, products were quantified by
high performance anion exchange chromatography with pulsed
amperometric detection (HPAEC-PAD). The kinetic parameters
showed a reduced hydrolytic activity of NXG1-ΔYNIIG compared to
NXG1 (Paper 2, Figure 3 and Table 3), while the
glycosyltransferase activity had increased.
The structure of NXG1 reveals the typical GH16 β-sandwich
fold (Paper 2, Figure 5) with a surprisingly high overall similarity
to PttXET16-34 (Paper 2, Figure 6a and 6b; PttXET16-34 structure
is described in Paper 3). Superimposed structures result in an
RMSD of 1.35 Å (241 Cα atoms) between PttXET16-34 (PDB id
1UN1) and NXG1, and a RMSD of 1.18 Å (221 Cα atoms) between
PttXET16-34 and NXG2. Both NXG variants contain the helical
extension of the N-terminus which is stabilized by two conserved
disulfide bonds, typical for members of the XTH gene family, while
it lacks the N-glycosylation in close proximity to the active site
(Paper 3), known to be present in most of the XTH gene family
members (Campbell and Braam, 1999a; Yokoyama et al., 2004).
Martin J. Baumann
38
The primary sequences of the NXG isoforms contain three
inserts when compared with the PttXET16-34 sequence. NXG1
has 3 inserts of 8,3, and 5 residues long, (Paper 3 supplemental
sequence alignment). Two of the inserts may interact with the
substrate since they are located at the substrate binding cleft.
The first insert belongs to the Loop extending from residue N84 to
residue D91 (Loop1, insert Y88PG90). It is situated directly
opposite to the negative subsites. The second insert is part of
Loop 2 (residue E117 to G126, insert Y122NIIG126) and is located
opposite to the positive subsites. This Loop was observed in three
different conformations in the 3D structure, indicating a high
flexibility. When superimposed with the XLLG-PttXET16-34
complex (PDB id 1UMZ) structure, I125 overlaps with Glc in the
+1 binding site. However this might not represent a determinant
for hydrolytic activity versus transglycosylating activity, since
xyloglucan hydrolysis requires substrate binding in the positive
subsites as well as in the negative subsites. Further work is
needed to understand this detail. The clash indicated by the
protein crystal structure could be resolved by protein flexibility.
Altogether the structural data in connection with the kinetic data
indicate that the extension of Loop 2 in NXG1 is one of the
determinants for transglycosylation activity.
Genes of the XTH family have been divided into subgroups
by several authors. In most cases three subgroups have been
recognized, and subgroup III was predicted to have exclusively
hydrolytic activity (Campbell and Braam, 1999a; Yokoyama et al.,
2004). A large number of sequences is currently available from
genome projects: 29 sequences from rice, 33 sequences from
Arabidopsis thaliana and 36 sequences from Populus trichocarpa.
Additional full length sequences are available from plant EST
projects: 13 sequences from hybrid aspen and 16 sequences from
Results and Discussion
39
tomato. Several single sequences from other species were also
included in our sequence analyses (supplemental Figure S5,
supplemental Figure S6). The bootstrap values indicate that the
previously known Group I and II are not clearly distinguishable,
several smaller Groups with high bootstrap values were observed
(supplemental Figure S6). All currently characterized proteins
from Group I and II have been shown to be transglycosylases, and
no clearly hydrolytic enzyme has been found in these Groups
(supplemental Table S1). Most notably Group III was shown to be
clearly divided into two subgroubs, Group III-A and III-B. A Loop
extension of three aminoacids of Loop1 is present in both
subgroups. The division between subgroup III-A and III-B
coincides with the length of the extension of Loop 2, Group III-A
has an extension of five aminoacids and Group III-B has only an
extension of 3 aminoacids when compared to sequences of Groups
I and II. Two enzymes from subgroup III-B have been currently
characterized (SlXTH5 and AtXTH2717), they show predominant
transglycosylating activity, with no detectable hydrolytic activity
(Campbell and Braam, 1999b; Van Sandt et al., 2006). This
indicates that the extension of Loop 1 does not influence the ratio
of hydrolytic to transglycosylating activity. NXG1 is currently the
only characterized protein from subgroup III-A, it shows clear
hydrolytic activity. The truncation of Loop 2 by five amino acids
(mutant NXG1-ΔYNIIG, Paper 2) does increase the
transglycosylation activity and reduces the hydrolytic activity. It
is clear that Loop 2 is a key structural change influencing the ratio
of transglycosylation activity versus hydrolytic activity. However a
number of other small not yet identified changes may influence
the fate of the glycosyl enzyme intermediate (Paper 2, Figure 1).
17 AtXTH27 was called previously EXGT-A3.
Martin J. Baumann
40
Several XTH genes from subgroup III-A have been analyzed
by expression profiling. NXG1 homologes are expressed during
fruit ripening of litchi (Lu et al., 2006) and grape (Ishimaru and
Kobayashi, 2002). Two Arabidopsis XTH gene products are
predicted to have a role in morphogenesis (AtXTH31) or being
expressed in the shoot apex (AtXTH32) (Becnel et al., 2006). The
gene products of Group III-A, are presumably hydrolytic as NXG1,
the expression data indicate that hydrolytic activity might be
required for fruit ripening and in fast developing tissues.
The divergent activities in the XTH gene family raise the
question of the origin of these activities. In our phylogenetic tree
(supplemental Figure S6) both main branches (Group I/II) contain
predominant XETs, while Xyloglucan hydrolases only appear in
Group III-A. Furthermore Group III-A is the least divergent from
all Groups, the pairwise identity is between 60 and 70%. The
typical structural characteristic which distinguishes the XTH gene
family from GH16 is the C-terminal extension. It is present in
XETs as well as in the xyloglucan hydrolases. This indicates that
xyloglucan hydrolases are closer related to the XETs than to the
other hydrolytic members of GH16. It is therefore likely that the
xyloglucan hydrolases have emerged from the XETs.
6.3. PttXET16-34 structure PttXET16A (this name is used in paper 3 and 5) has recently
been renamed to PttXET16-34 (Geisler-Lee et al., 2006). The
structure of PttXET16-34 has been solved and it is the typical
GH16 β-sandwich (Paper 3, Figure 1A, PDB id for apo-structure:
1UN1, PDB id for complex structure: 1UMZ). GH16 and GH7
have the same overall fold and are grouped into clan B. In spite
of low sequence similarity within this clan, the positions of the
catalytic residues (nucleophile, helper and acid/base) match
Results and Discussion
41
exactly in a structural overlay (Paper 3 Figure 1D). Apart from
this only few aromatic residues positioned along the substrate
binding site have matching positions (Paper 3, Figure 2B and C).
PttXET16-34 has an N-glycosylation site, which is glycosylated
when the protein is expressed by P. pastoris. It was shown that
de-glycosylation (enzymatic or by mutagenesis) does not affect
activity, but it seems to reduce protein solubility (Kallas et al.,
2005). Highly ordered electron density for two N-acetyl
glucosamine residues and one mannose residue was observed. A
network of no less than seven hydrogen bonds to several residues
of the protein stabilizes three sugar residues in the protein crystal
(Paper 3, Figure 1C).
In order to study the interaction of the protein with the
substrate in detail, co-crystallization with small, well defined
xyloglucan oligosaccharides (XGO) was attempted. A complex
structure was obtained with a 2-choro-nitrophenol-XLLG or XLLG
bound in the positive subsites (acceptor site). Clearly ordered
electron density was observed for an oligosaccharide equivalent to
XLG (Paper 3 Figure 3C and Figure 4), indicating that the rest of
the oligosaccharide is not in close contact to the protein. The
three β(1→4) linked glucose units are in a linear conformation,
with the two observed α(1→6) bound xylose units pointing
outwards to different sides. The inferred nucleophile E89 is in
close contact with the 3’ and 4’ OH-group of the terminal glucose
on the non reducing end of the oligosaccharide (Paper 3 Figure 3B
and C).
The kinetic analysis showed that PttXET16-34 does not use
XLLG-CNP as a donor substrate, but as an acceptor in the
presence of high Mr xyloglucan (Paper 1, Figure 5). This indicates
that sugars are required to bind in the positive subsides for
catalysis to occur.
Martin J. Baumann
42
6.3.1. PttXET16-35 characterization
Expression studies indicate that PttXET16-3418 (Paper 3) and
PttXET16-35 are both up-regulated in cambium, and during cell
expansion and xylogenesis. PttXET16-35 has higher expression
levels in phloem and tension wood, and it is up-regulated in leaf
and shoot, whereas PttXET16-34 is down-regulated in these
tissues. The study indicates that PttXET16-35 is regulated
individually and tissue-specifically, like other members of the XTH-
gene family.
PttXET16-35 is highly similar to PttXET16-34 (53% identical,
70% similarity). Two outstanding differences make the protein
interesting: The sequence around the catalytic residues is atypical
(Paper 4, Figure 1) and the protein has two glycosylation sites
(Paper 4, Figure 1). The first site is conserved and found in
PttXET16-34 (6.3), situated in close proximity to the catalytic
residues; the second one is at the amino terminal region, situated
on the Loop connecting strand β3 (according to PttXET16-34
structure) and strand β4, presumably pointing towards the
surrounding solution. In the PttXET16-35/N29K mutant, this
glycosylation site was removed.
PttXET16-35 and its mutant were difficult to purify with
classical protein column-chromatography. Therefore, a novel
affinity chromatographic method was employed which yielded
small amounts of pure protein. The mutant PttXET16-35/N29K
was obtained in larger amounts and was used for subsequent
characterization of the enzyme.
The activity measurements reveal a somewhat lower specific
activity than PttXET16-34. The pH optimum was slightly shifted
towards the acidic side (Paper 4, Figure 2) and the melting
temperatures (Paper 4, table 2) were clearly lower. No hydrolytic
18 Previously called PttXET16A
Results and Discussion
43
activity was observed of either PttXET16-35 or PttXET16-35/N29K.
Similar to PttXET16-34, deglycosylation of PttXET16-35/N29K by
the endoglycosidase endoH did not affect the activity.
6.4. Fiber surface modification Fiber surface modification has gained increasing importance
in industrial product development. To meet the requirements of
the printing industry, the almost pure cellulosic fibers of paper
must be combined with various kinds of coatings and filler
materials during the paper making process. Cellulose fibers are a
raw material with excellent physical properties (high specific
stiffness and tensile strength), but the surface offers very little
chemical reactivity. Most of the hydroxyl groups (both primary
and secondary) on the cellulose fiber form tight interactions in the
crystal lattice and are therefore slow to undergo chemical
reactions. Chemical modifications of the hydroxyl groups often
result in a drastic loss of strength of the cellulose fiber, due to
crystal disintegration.
Xyloglucan binds with high affinity to cellulose surfaces
without disrupting fiber integrity (Vincken et al., 1995; Mishima et
al., 1998; de Lima and Buckeridge, 2001). Xyloglucan
oligosaccharides are small (the Glc4 units XXXG, XLXG, XXLG or
XLLG have a molecular masses between 1062 g/mol to 1386
g/mol) and are well-defined. A large variety of analytical tools is
readily applicable (TLC, high resolution mass-spectrography, NMR
and CD). The reducing end of the oligosaccharides has a relative
high reactivity under mild conditions. XGOs can be transferred to
high
Martin J. Baumann
44
XG + XGO-RXET
XG-R
R=N
H-FITC R
=NH 2
FITC
R=OBA
UV light
R=N
HC
O(C
H2 )3 S
H
DTT
MTS
-Rhodam
ine
R=NH-biotin
1. SA-AP2. BCIP/NBT
R=A
TRP
initi
ator
met
hyl
met
hacr
ylat
e
OHO O
CO2H
NHS
HN
O(CH3CH2)2N N(CH 2CH3)2
S
SO3
O
NH
O
S
HN
O
S
NH HN
O
H H
SH
O
HO
O
HO2C
NH
SHN
H2N
S
HN
-
-
O
O
3
3
S
S
NH2
HN
-
-
O
O
3
3
S
S
NH2
NH
OO
O
O
Br
O
O
Br
MeO2C
n
Figure 9. Schematic representation of the XET-technology, functionalized xyloglucan (XG-R) is made by XET from xyloglucan and functionalized XGOs. The functional groups can be: fluorescein, for detection; an amino-group subsequently reacted with fluorescein-isothiocyanate; an optical brightener; biotin for subsequent reaction with streptavidin; a thiol group, subsequently reacted with Rhodamine; or an initiator for subsequent RTRP-polymerization. Figure adapted from (Zhou et al., 2006b)
Mr xyloglucan by the transglycosylating XET. As a result, the
synthetic chemistry can be carried out on small well-defined
XGOs, and later introduced to the polymeric xyloglucan by an
enzymatic reaction.
Results and Discussion
45
The aminoalditol derivative of an XGO (XGO-NH2, Paper 5
Figure 2B) serves as a key intermediate for biomimetic fiber
modification. As described above, XGO-NH2 can be incorporated
into high molecular mass xyloglucan using the transglycosylation
activity of PttXET16-34. The product of this reaction is aminated
xyloglucan (XG-NH2). The enzyme allows on one hand, small
well-defined XGOs to be used in chemical reactions, while on the
other hand, permitting modified high molecular mass xyloglucan
to be used for fiber modification. Aminated xyloglucan can be
readily adsorbed to cellulosic surfaces (Paper 5, Figure 2A upper
pathway). The high intrinsic reactivity of the amino group allows
fiber surface modifications under very mild conditions.
Alternatively, XGO-NH2 can be further derivatized with a
number of different functional groups and the reaction product can
be incorporated into xyloglucan by PttXET16-34 (Paper 5, Figure
1A lower pathway). This pathway has the advantage that all
chemical reactions are done in solution and no reactant comes in
contact with the cellulose. Further, the modified products can be
easily isolated and characterized by standard methods such as
TLC, NMR and high resolution MS. Here, XGO-biotin was used as
an example, showing that it is possible to bind the strepavidin-
alkaline phosphatase conjugate (Paper 5, Figure 1B structure 5).
These examples (Figure 9) serve as a proof-of-principle for a
readily applicable technique for a mild and biomimetic fiber
surface modification method. Future work will expand the
spectrum of functional groups incorporated via PttXET16-34
modified xyloglucan.
This method has been further expanded by a number of
subsequent publications (Zhou et al., 2005; Lönnberg et al.,
2006; Zhou et al., 2006b), which can be found in the beginning of
this thesis under additional publications.
46
7. Concluding remarks and future perspectives The use of enzymes to replace chemicals in industrial
processes is of significant importance for a more sustainable
development in the future. Detailed enzymatic studies at the
molecular level provide the fundamental understanding required
to use and engineer proteins towards new applications.
Non-NDP-sugar glycosyltransferases are a new class of very
attractive enzymes for potential industrial applications, even
though they are not as well studied as hydrolases. In particular
the lack of hydrolytic activity is poorly understood. A comparative
study of mixed function enzymes like NXG1 and XETs will provide
insight into the structural requirements of transglycosylation.
Structural studies of mutants together with kinetic studies will
help to identify key interactions between protein and substrate.
The novel biomimetic fiber modification technique (XET-
technology) here developed is a good example how basic
enzymological studies can lead to novel products. In order to
satisfy industrial demands feasible production and purification
methods for the relevant enzymes have to be developed, and
moreover, methods for simple and reliable kinetic
characterization. Xyloglucan-active enzymes and
transglycosylases, like XETs in particular, are challenging study
objects since the utilized substrate is polymeric and branched.
However, their interesting potential for industrial applications
motivates further efforts in the field of xyloglucan-active enzymes.
47
8. Acknowledgements Many have contributed to this thesis, first, Tuula Teeri for
accepting me as a PhD student, Harry Brumer as an enthusiastic
supervisor, and for actually offering me the position and helping
me with many small important things in a foreign country. I was
lucky to be working with a long list of excellent Postdocs, they
were Qi Zhou (in the early days of fiber modification), Lionel
Greffe (organic chemistry and flash columns), Kathleen Piens
(protein expression and PttXET16-34 mutants) and Farid M.
Ibatullin (remaking and expanding on the theme of aryl-XGOs).
I would also like to thank Mirjam Czjzek for the collaboration
on the amazingly structure work of NXG1.
Then my colleagues working with me on different projects;
first of all Åsa Kallas, making the NXG1 project go further than to
mRNA level, and especially for her patience to actually speak
Swedish with me. The previous diploma workers and now
colleagues, Jens Eklöf and Fredrika Gullfot, making me feel as if I
had six left19 hands. And my colleagues working around me are
thanked for the patience and help (in alphabetical order): Alex,
Anders W., Christian, Henrik, Johanna, Lage, Maria H., Nomchit,
Soraya and Ulla.
The financial support from the Swedish Forest Industries
Federation (Skogsindustrierna) 20 through the “Biofibre Materials
Centre”21 is gratefully acknowledged.
19 “left” was inserted here by Fredrika herself, very special thanks for careful proofreading and the translation of the abstract. 20 More information available http://www.skogsindustrierna.org 21 More information available at www.bimac.se
48
9. List of abbreviations CD circular dichroism
DTKP de-oiled tamarind kernel powder
endoH Endoglycosidase H
ESI-TOF MS electro-spray ionization time of flight
mass spectroscopy
F α-L-Fucp(1→2)-β−D-Galp(1→2)
G Glcp
GH glycosyl hydrolase family
GT glycosyl transferase family
HPAEC-PAD high performance anion exchange
chromatography with pulsed
amperometric detection
L β−D-Galp(1→2)- α-D-Xylp(1→6)-Glcp
MS mass spectroscopy
NMR nuclear magnetic resonance
S α-D-Araf(1→2)- α-D-Xylp(1→6)-Glcp
TLC thin layer chromatography
TKP tamarind kernel powder
X α-D-Xylp(1→6)-Glcp- α-D-Xylp(1→6)-Glcp
XEH xyloglucan endohydrolase (EC 3.2.1.151)
XET xyloglucan-endotransglycosylase (EC 2.4.1.204)
XG xyloglucan
XGase xyloglucan hydrolase
XGO xyloglucan oligosaccharide
XTH xyloglucan transferase/hydrolase gene
family
49
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