Xyloglucan-Active Enzymes: Properties, Structures and Applications

Xyloglucan-active enzymes: properties, structures and applications.

Martin J. Baumann Doctoral thesis

School of Biotechnology Royal Institute of Technology

Stockholm 2007

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

ii Abstract

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.

iii Sammanfattning

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.

iv Zusammenfassung

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.

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vi

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

vii

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.

viii Table of contents

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

1 Martin J. Baumann

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

2 Introduction

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)

3 Martin J. Baumann

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.

4 Introduction

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.

5 Martin J. Baumann

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).

Cell walls Glucuronoarabinoxylans Commelinoid Ferrulic acid (1→3), (1→4)-β-glucans (grasses) Monocotyledons Storage Fructan β(2→6)−fructans (grasses)

Flowering Cell walls Plants Non Commelinoid Xyloglucans Pectic Polysaccharides

Dicotyledons Storage Fructan β(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.

6 Xyloglucan is a cell wall polysaccharide

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).

7 Martin J. Baumann

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.

8 Xyloglucan is a cell wall polysaccharide

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

HO HO OH OH

O O HO O OH x O OH HO HO HO O HO O O O O HO O OH H O O HO HO HO O O HO OH O OH O O O HO OH y OH HO OH OH

n

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.

9 Martin J. Baumann

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

10 Xyloglucan is a cell wall polysaccharide

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

4 Bilberry (Vaccinium XXXG, XXUG, XXLG, XXFG, XLFG (Hilz et al., 2007) myrtillus L)

Black currant, (Ribes XXXG, XXFG, XLFG, XLXG , XXLG , XLLG (Hilz et al., 2006) nigrum L) (30:31:35:2:2:1)

Afzelia africana XXXG, XLXG, XXLG, XLLG (1.00:0.21:2.80:1.68) (Ren et al., 2004)

Tamarindus indica kernel XXXG, XLXG, XXLG, XLLG (Marry et al., 2003) powder (1.000:1.160:1.240:1.429)

Hymenaea courbaril XXXXG based subunits and Glc6 based subunits (Buckeridge et al., 1997; cotyledones Tine et al., 2003; Tine et al., 2006) Olive (olea europaea cv XXSG, XLSG, some of the arabinose residues are (Vierhuis et al., 2001) koroneiki) fruit pulp substituted with either 1 or 2 O-acetyl groups

Persimmon (Diospyros Glc:Xyl:Gal:Fuc (10.0:6.0:3.4:1.4) (Cutillas-Iturralde et al., kaki L.) fruit pulp 1998)

Apple (Malus malus L.) XXXG, XLXG, XXFG, XLFG (4:2:3:4) (Vincken et al., 1996a) fruit pulp (Spronk et al., 1997)

Secreted from Based on subunits XXGG (34%) and XXGGG (Sims et al., 1996) suspension culture of (27%), one (60%) or two (15%) terminal Araf Nicotiana plumbaginifolia residues attached at O-2 of the Xylp residues.

Potato (Solanum XXGG-type subunits (Vincken et al., 1996b) tuberosum var. Bintje)

Detarium senegalese XXXG, XLXG, XXLG, XLLG (1.00:0.30:5.60:6.20) (Wang et al., 1996) Gmelin cotyledons

Tamarindus indica XG from rapeseed: XXXG, XXLG, XLXG, XXFG, (York et al., 1990; xyloglucan, sycamore XLFG (11.5:1:3:9.4:16.9) Hisamatsu et al., 1991; extra-cellular XG from tamarind seed: XXXG, XXLG, XLXG, XLLG Hisamatsu et al., 1992; polysaccharide (Acer (1.4:3:1:5.4) York et al., 1993; York et pseudoplatanus) and al., 1995) rapeseed hulls (brassica naptus)

Suspension-cultured Wall XG: XLFG, XXFG, XXLG, XXXG (Hayashi and Takeda, poplar cells (0.58:1.76:0.54:1.0) 1994) Extra-cellular XG: XLFG, XXFG, XXLG, XXXG (0.53:2.08:0.65:1.0)

4 More than 20 oligosaccharides have been identified, only the most abundant (more than 10%) are listed here.

11 Martin J. Baumann

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)

12 Xyloglucan is a cell wall polysaccharide 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).

13 Martin J. Baumann

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

14 Xyloglucan is a cell wall polysaccharide 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

15 Martin J. Baumann

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 Reference and binding conditions

Tamarindus indica bleached chemical and mechanical wood pulps (Zhou et al., 2006a)

(Zykwinska et al., Tamarindus indica Avicel 2005)

Rubus fruticosus suspension cultures Primary cell wall, cotton, flax, bacterial cellulose, tuniciae, at different time points of growth rhizoclonium, quantification of unbound XG (Chambat et al., curve 2005)

Tamarindus indica Filter paper, binding at 20°C, in pure water, quantification (Brumer et al., 2004) of FITC labeled XG on the paper surface, or of remaining XG in solution

Phaseolus vilgaris L (common bean) Microcrystalline cellulose (Lima et al., 2004) C. langsdorfii, Tamarindus indica and Binding at 30°C, pH 6.0, 15 Min., quantification of H. courbaril remaining XG in solution

Phaseolus vilgaris L Microcrystalline cellulose, (de Lima and (common bean) Binding at 5-60°C, pH 2-8, 15 Min., quantification of Buckeridge, 2001) C. langsdorfii, remaining XG in solution Tropaeolum majus, Tamarindus indica and H. courbaril

Tamarindus indica oligosaccharides Avicel and cotton linters (Vincken et al., 1995) (1-11 repeating units and high MW) Annealing at 40°C pH 3 and 5.8, 6h, quantification of remaining XG in solution

Pea (pisum sativum L. Var. Alaska) Pea cellulose, (Baba et al., 1994) XG extracted and reconstituted, immuno-gold detection

Tamarindus indica and Amorphous cellulose (PASC) (Hayashi et al., Pisum sativum annealing at 30°C –120°C, carbohydrate quantification 1994a) high MW down to 1000 KDa after binding and re-extraction of XG from formed complex.

Tamarindus indica, oligosaccharides Amorphous cellulose, microcrystalline cellulose, (Hayashi et al., (DP8-12 and 3000-64) 5 annealing at 120°C 10 Min., pH 5.5, 3H labeled 1994a) pisum sativum (DP 150) 4 and cello scintillation counting of bound radioactivity. oligosaccharides (DP5-6) 4 Binding isotherms of XGO and cello oligosaccharides

Tamarindus indica, oligosaccharides Amorphous cellulose, microcrystalline cellulose, filter (Hayashi et al., (DP3-12) 4 and cellooligosaccharides paper, 1994b) (DP2-6) 1 annealing at 120°C 10 Min., pH 5.5, 3H labeled scintillation counting of bound radioactivity. Binding isotherms of XGO and cello oligosaccharides

Pine (pinus pinaster) from seedlings Commercial cellulose (CF-11 or CF-41 Whatman), pine (Acebes et al., 1993) 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

Pea (pisum sativum) Pea cellulose, Acetobacter xylinum cellulose, Valonia (Hayashi et al., 1987) ventricosa cellulose, Binding at pH 5, 40°C, 6h, radioactive detection (125I) or fluorescent labeled XG, competition experiments with other hemicelluloses

Pea (pisum sativum L. Var. Alaska), Pea cellulose (cell wall ghosts) (Hayashi and Tamarindus indica and cellopentaose Binding visualization with fluorescein labeled fucose Maclachlan, 1984) binding lectin (U. europeus), radioactive label (3H) and iodine staining of XG-cellulose complex – and reconstitution in vitro

5 Number of 1,4-β-glycosyl units

16 Xyloglucan is a cell wall polysaccharide

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).

17 Martin J. Baumann

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/

18 Xyloglucan-active enzymes 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.

19 Martin J. Baumann

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 (Yaoi et al., 2004) cellobiohydrolase 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)

20 Xyloglucan-active enzymes

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

21 Martin J. Baumann

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.

Golgi membrane

NDP

NDP- NDP- NDP GT Golgi lumen CSL CSL Cytosol

NDP- NDP

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).

22 Xyloglucan-active enzymes

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.

23 Martin J. Baumann

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)

OH OH OH HO O H O O O O HO O HO O HO O HO O HO O R R H OH HO OH OH O R O H OH OH H H O O O O HO O

Figure 6. The inverting transglycosylation mechanism, adapted from Withers (2001), the distance between the general base and the general acid is typically 11 Å.

24 Xyloglucan-active enzymes

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.

25 Martin J. Baumann

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).

OH OH OH HO O O O HO O HO O HO O HO O H HO HO HO OH O R O OH OH H OH O O O O O O

Figure 7. The retaining hydrolysis mechanism, adapted from Withers (2001), the distance between nucleophile and acid/base is typically 5.5 Å.

26 Xyloglucan-active enzymes

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

27 Martin J. Baumann

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.

28 Xyloglucan-active enzymes 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).

OH OH OH HO O O O HO O HO O HO O HO O H HO HO HO O R O O R' OH OH R' OH O O O O O O

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

29 Martin J. Baumann

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 Reaction Enzyme activity Selected References family mechanism 2.4.1.19 13 retaining cyclomaltodextrin glucanotransferase (Lawson et al., 1994) 2.4.1.18 branching enzyme (Abad et al., 2002) 2.4.1.25 4-α-glucanotransferase (Roujeinikova et al., 2002) 2.4.1.4 Amylosucrase (Mirza et al., 2001) 2.4.1.7 sucrose phosphorylase (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 32 retaining sucrose:sucrose 1-fructosyl transferase (Vijn et al., 1998) 2.4.1.100 fructan fructan 1-fructosyltransferase (van der Meer et al., 1998)

2.4.1.- 33 retaining trans-sialidase (Amaya et al., 2004)

2.4.1.67 36 retaining stachyose synthase (Peterbauer et al., 2002) 2.4.1.82 raffinose synthase

2.4.1.25 57 retaining 4-α-glucanotransferase (Laderman et al., 1993)

2.4.1.- 66 retaining cycloisomaltooligosaccharide glucano- (Oguma et al., 1994) transferase

2.4.1.10 6813 retaining levansucrase (Meng and Futterer, 2003) 2.4.1.9 inulosucrase (van Hijum et al., 2002)

2.4.1.5 7014 retaining Dextransucrase (Kang et al., 2005) 2.4.1.140 Alternansucrase (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.

30 Xyloglucan-active enzymes

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

31 Martin J. Baumann 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.

32 Aims of the present investigation

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.

33 Martin J. Baumann

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 source activity PDB id PDB id family apo-structure ligand-structure

CtXGH74A GH74 Clostridium thermocellum Xyloglucanase 2CN2 2CN3 (3.2.1.151)

NXG1 GH16 Tropaeolum majus Xyloglucanase /XET In preparation -

(2.4.1.207/ 3.2.1.151)

PttXET16-34 GH16 Populus tremula x XET 1UN1 1UMZ tremuloides (2.4.1.207)

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

34 Results and Discussion 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

35 Martin J. Baumann

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,

36 Results and Discussion

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).

37 Martin J. Baumann

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

38 Results and Discussion 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 AtXTH27 17 ), 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.

39 Martin J. Baumann

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

40 Results and Discussion 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.

41 Martin J. Baumann

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

42 Results and Discussion 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

43 Martin J. Baumann

HO

S HN N O H HO C HO O O 2 O

CO2H NH2

FITC -O S 3 S NH

H N - HN 2 NH2 SO3

-O S HN 3

- SO3 HN

R=NH-FITC UV light

2

R=NH

R=OBA XET O S XG + XGO-R XG-R HN R=NH-biotin H H R=NHCO(CH HN NH or

O

2 ) 1. SA-AP 3 SH R=ATRP initiat 2. BCIP/NBT O

NH O Br O SH O

MTS-Rhodamine (CH3CH2)2N O N(CH 2CH3)2

Br DTT methyl n SO3 O methacrylate MeO2C O O S O

S NH S

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.

44 Results and Discussion

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.

45

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.

46

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

47

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

48

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