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Plant and Microbial Xyloglucanases Plant and microbial xyloglucanases: Function, Structure and Phylogeny Jens Eklöf Doctoral thesis Royal Institute of Technology School of Biotechnology Division of Glycoscience Stockholm 2011 Plant and microbial xyloglucanases: Function, Structure and Phylogeny ISBN 978‐91‐7415‐932‐5 ISSN 1654‐2312 Trita‐BIO Report 2011:7 ©Jens Eklöf, Stockholm 2011 Universitetsservice US AB, Stockholm ii Jens Eklöf Jens Eklöf (2011): Plant and microbial xyloglucanases: Function, Structure and Phylogeny School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden Abstract In this thesis, enzymes acting on the primary cell wall hemicellulose xyloglucan are studied. Xyloglucans are ubiquitous in land plants which make them an important polysaccharide to utilise for microbes and a potentially interesting raw material for various industries. The function of xyloglucans in plants is mainly to improve primary cell wall characteristics by coating and tethering cellulose microfibrils together. Some plants also utilise xyloglucans as storage polysaccharides in their seeds. In microbes, a variety of different enzymes for degrading xyloglucans have been found. In this thesis, the structure‐function relationship of three different microbial endo‐xyloglucanases from glycoside hydrolase families 5, 12 and 44 are probed and reveal details of the natural diversity found in xyloglucanases. Hopefully, a better understanding of how xyloglucanases recognise and degrade their substrate can lead to improved saccharification processes of plant matter, finding uses in for example biofuel production. In plants, xyloglucans are modified in muro by the xyloglucan transglycosylase/hydrolase (XTH) gene products. Interestingly, closely related XTH gene products catalyse either transglycosylation (XET activity) or hydrolysis (XEH activity) with dramatically different effects on xyloglucan and on cell wall characteristics. The strict transglycosylases transfer xyloglucan segments between individual xyloglucan molecules while the hydrolases degrade xyloglucan into oligosaccharides. Here, we describe and determine, a major determinant of transglycosylation versus hydrolysis in XTH gene products by solving and comparing the first 3D structure of an XEH, Tm‐NXG1 and a XET, PttXET16‐ 34. The XEH activity was hypothesised, and later confirmed to be restricted to subset of the XTH gene products. The in situ localisation of XEH activity in roots and hypocotyls of Arabidopsis was also visualised for the first time. Furthermore, an evolutionary scheme for how XTH gene products developed from bacterial β‐1,3;1,4 glucanases was also presented based on the characterisation of a novel plant endo‐glucanase, PtEG16‐1. The EG16s are proposed to predate XTH gene products and are with activity on both xyloglucan and β‐1,3;1,4 glucans an “intermediate” in the evolution from β‐ 1,3;1,4 glucanases to XTH gene products. Keywords, xyloglucan, XTH, XET, XEH, xyloglucanase, plant cell wall, phylogeny, GH16, endo‐glucanase iii Plant and microbial xyloglucanases: Function, Structure and Phylogeny Sammanfattning I den här avhandlingen undersöks enzymer som modifierar hemicellulosan xyloglukan. Xyloglukaner finns i alla landlevande växter där de och binder till, täcker och korslänkar cellulosafibriller i den primära cellväggen. Funktionen av xyloglukan är att spatialt separera cellulosafibriller, men ändå behålla cellväggens styrka. I cellväggen modifieras xyloglukan av enzymer från xyloglukan transglykosylas/hydrolas (XTH) genfamiljen. Dessa närbesläktade enzymer, som antingen katalyserar transglykosylering (XET‐aktivitet) eller hydrolys (XEH‐aktivitet) av xyloglukan har vitt skilda effekter på xyloglukan och därför också på cellväggens egenskaper. Strikta transglykosylaser utbyter xyloglukanbitar mellan olika xyloglukanmolekyler medan hydrolaserna klyver ner xyloglukan till oligosackarider. Mikrober som lever på och bryter ner växtmaterial och således också xyloglukan har utvecklat olika typer av xyloglukanaser. Tre mikrobiella xyloglukanaser, från glykosidhydrolasfamiljerna 5, 12 och 44, vars 3D‐strukturer och biokemiska karaktärisering demonstreras i denna avhandling påvisar en del av den naturliga variationen av xyloglukanaser. En bättre förståelse av dessa enzymer och hur de känner igen sina substrat kan förhoppningsvis leda till förbättrad nedbrytning av växtmaterial för t.ex. biobränslen. Även växter är i behov av xyloglukanaser. Den första 3D‐strukturen av en XEH, Tm‐NXG1 kopplar en strukturell skillnad, mellan transglykosylaser och hydrolaser, till en funktionell skillnad, och kan därigenom lokalisera hydrolas aktivitet i XTH‐genprodukter till några väl avgränsade enzymer. Vävnadsspecificiteten i XEH‐aktivitet hos modellorganismen backtrav, Arabidopsis thaliana, demonstreras även för första gången. Dessutom, föreslås hur XTH‐genprodukterna har utvecklats från bakteriella β‐1,3;1,4‐glukanaser genom upptäckten och karaktäriseringen av en ny typ av växt‐ endo‐glukanas, PtEG16‐1. PtEG16‐1 indelas i en ny grupp av växtenzymer som tros vara äldre än XTH‐ genprodukter och har både β‐1,3;1,4‐glukanas aktivitet och xyloglukanas aktivitet samt har likheter i aminosyrasekvens med både bakteriella β‐1,3;1,4‐glukanaser och XTH‐genprodukter. iv Jens Eklöf List of publications included in thesis I Ariza A*, Eklöf JM*, Spadiut O*, Offen WA, Roberts SM, Wilson KS, Brumer H, Davies GJ Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 44. (Manuscript) II Gloster TM, Ibatullin FM, Macauley K, Eklöf JM, Roberts S, Turkenburg JP, Bjørnvad ME, Jørgensen PL, Danielsen S, Johansen KS, Borchert TV, Wilson KS, Brumer H, Davies GJ (2007) Characterization and Three‐dimensional Structures of Two Distinct Bacterial Xyloglucanases from Families GH5 and GH12. J. Biol. Chem. 282: 19177‐19189 III Baumann MJ, Eklöf JM, Michel G, Kallas AM, Teeri TT, Czjzek M, Brumer H, III (2007) Structural Evidence for the Evolution of Xyloglucanase Activity from Xyloglucan Endo‐ Transglycosylases: Biological Implications for Cell Wall Metabolism. Plant Cell 19: 1947‐1963 IV Kaewthai N*, Eklöf JM*, Gendre D*, Ibatullin FM, Ezcurra I, Bhalerao RP, Brumer H Group III‐ A XTH Genes Encode Predominant Xyloglucan endo‐hydrolases Active in Expanding Tissues of Arabidopsis thaliana. (Manuscript) V Maris A, Kaewthai N*, Eklöf JM*, Miller JG, Brumer H, Fry SC, Verbelen JP, Vissenberg K (2011) Differences in Enzymic Properties of Five Recombinant Xyloglucan endotransglucosylase/hydrolase (XTH) Proteins of Arabidopsis thaliana. J. Exp. Bot. 62: 261‐ 271 VI Eklöf JM, Brumer H, An endo β‐1,4 glucanse, PtEG16‐1 from black cottonwood (Populus trichocarpa) represents an evolutionary link between bacterial lichenases and XTH gene products. (Manuscript) *Authors contributed equally to the work v Plant and microbial xyloglucanases: Function, Structure and Phylogeny Author’s contributions Paper I: Performed part of the experimental work primarily on polysaccharides and inhibition studies. I also made significant contributions to the writing of the paper. Paper II: Performed the experimental work concerning polysaccharide specificity and pH optimum. Paper III: Took part in almost all parts of the paper except the crystallography. The experimental in vitro work was done together with Dr. Martin Baumann and the phylogenetic analyses were performed together with Dr. Gurvan Michel. Paper IV: Performed the production, purification and characterisation AtXTH31 in vitro. Paper V: Performed the production, purification and characterisation AtXTH13 in vitro and contributed in the writing of the paper. Paper VI: This work was performed by me; from idea, to cloning and characterisation and phylogenetic analyses. vi Jens Eklöf Related publications not included in this thesis 1. Eklöf JM, Brumer H (2010) The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling. Plant Physiol. 153: 456‐466 (Included, in part as a chapter in the introduction.) 2. Eklöf JM*, Tan TC*, Divne C, Brumer H (2009) The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 8. Proteins 76: 1029‐1036 3. Mark P, Baumann MJ, Eklöf JM, Gullfot F, Michel G, Kallas AM, Teeri TT, Brumer H, Czjzek M (2009) Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo‐transglycosylases and endo‐hydrolases. Proteins: Struct. Funct. Bioinf. 75: 820‐836 4. Nordgren N, Eklöf JM, Zhou Q, Brumer H, Rutland MW (2008) Top‐down grafting of xyloglucan to gold monitored by QCM‐D and AFM: Enzymatic activity and interactions with cellulose. Biomacromolecules 9: 942‐948 *Authors contributed equally to the work vii Plant and microbial xyloglucanases: Function, Structure and Phylogeny Content Introduction ............................................................................................................................................. 1 Plants & Man ....................................................................................................................................... 2 The Origin & Early Evolution of Plants ................................................................................................ 2 The Embryophytes ............................................................................................................................... 3 The Plant Cell Wall ..............................................................................................................................
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