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Xyloglucan-active enzymes: properties, structures and applications
KTH, School of Biotechnology (BIO).
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [sv]

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.

Abstract [de]

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.

Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: KTH , 2007. , viii, 59 p.
Keyword [en]
xyloglucan, xyloglucan endo-transglycosylase/hydrolase, xyloglucan endo-hydrolase, xyloglucan endo-transglycosylase, XET, xyloglucan oligosaccharides, synthesis, carbohydrate, cellulose, crystal structure, fiber, surface modification, Populus tremula x Populus tremuloides, Hybrid aspen, nasturtium, NXG
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-4314ISBN: 978-91-7178-596-1 (print)OAI: oai:DiVA.org:kth-4314DiVA: diva2:11761
Public defence
2007-04-13, SAL FD5, AlbaNova, Roslagstullbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100624Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2012-02-23Bibliographically approved
List of papers
1. Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation
Open this publication in new window or tab >>Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 34, 24922-24933 p.Article in journal (Refereed) Published
Abstract [en]

The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.

Keyword
END-SPECIFIC CELLOBIOHYDROLASE; PLANT-CELL WALL; GLYCOSYL HYDROLASES; CELLULOSE SURFACES; EXPRESSION; CLONING; CLASSIFICATION; ENDOGLUCANASE; BIOSYNTHESIS; PURIFICATION
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6910 (URN)10.1074/jbc.M603583200 (DOI)000239847800085 ()2-s2.0-33747635388 (Scopus ID)
Note
QC 20100624Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2017-12-14Bibliographically approved
2. Structural analysis of nasturtium NXG reveals the evolution of GH16 xyloglucanase activity from XETs: biological implications for cell wall metabolism
Open this publication in new window or tab >>Structural analysis of nasturtium NXG reveals the evolution of GH16 xyloglucanase activity from XETs: biological implications for cell wall metabolism
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(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6911 (URN)
Note
QC 20100624Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2010-06-24Bibliographically approved
3. Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of the transglycosylation acceptor binding
Open this publication in new window or tab >>Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of the transglycosylation acceptor binding
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2004 (English)In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 16, no 4, 874-886 p.Article in journal (Refereed) Published
Abstract [en]

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure-function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-Angstrom resolution. Even though the overall structure of PttXET16A is a curved beta-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional beta-strand and a short alpha-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transgilycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.

Keyword
PLANT-CELL WALL; TRICHODERMA-REESEI; CELLOBIOHYDROLASE-I; MACROMOLECULAR STRUCTURES; GLYCOSIDE HYDROLASES; ARABIDOPSIS ROOTS; DONOR SUBSTRATE; ACTIVE-SITE; OLIGOSACCHARIDES; NOMENCLATURE
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6912 (URN)10.1105/tpc.020065 (DOI)000220731900010 ()2-s2.0-1842813472 (Scopus ID)
Note
QC 20100624Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2017-12-14Bibliographically approved
4. Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloides
Open this publication in new window or tab >>Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloides
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(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5705 (URN)
Note
QC 20100624Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved
5. Activation of crystalline cellulose surfaces though the chemoenzymatic modification of xyloglucan
Open this publication in new window or tab >>Activation of crystalline cellulose surfaces though the chemoenzymatic modification of xyloglucan
Show others...
2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 18, 5715-1721 p.Article in journal (Refereed) Published
Abstract [en]

Cellulose constitutes an important raw material for many industries. However, the superb load-bearing properties of cellulose are accompanied by poor chemical reactivity. The hydroxyl groups on cellulose surfaces can be reacted but usually not without loss of fiber integrity and strength. Here, we describe a novel chemoenzymatic approach for the efficient incorporation of chemical functionality onto cellulose surfaces. The modification is brought about by using a transglycosylating enzyme, xyloglucan endotranglycosylase, to join chemically modified xyloglucan oligosaccharides to xyloglucan, which has a naturally high affinity to cellulose. Binding of the chemically modified hemicellulose molecules can thus be used to attach a wide variety of chemical moieties without disruption of the individual fiber or fiber matrix.

Keyword
PLANT-CELL WALLS; NATURAL FIBERS; ENDOTRANSGLYCOSYLASE; TAMARIND; OLIGOSACCHARIDES; PERFORMANCE; COMPOSITES; SEEDS; SISAL
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6914 (URN)10.1021/ja0316770 (DOI)000221268400021 ()2-s2.0-2442542178 (Scopus ID)
Note
QC 20100624Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2017-12-14Bibliographically approved

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