xyloglucan-active enzymes: properties, structures …11761/fulltext01.pdf · xyloglucan-active...

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.

vii

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.


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


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


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)


13

polymer is not fucosylated (York et al., 1993). A number of

Indian companies specialize in supplying TKP; extracted on


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


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


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)


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).


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.


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.


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.


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.


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,


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


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


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


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.


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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