Kinetic analyses of retaining endo-(xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides
2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 29, 7762-7769 p.Article in journal (Refereed) Published
A library of phenyl beta-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on GlC(4) backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by beta-galactosidase treament. Subsequent per-O-acetylation, alpha-bromination, phase-transfer glycosylation, and Zemplen deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pK(a) values. Kinetic and product analysis of the action of the archetypal plant endo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl-enzyme intermediate was rate-limiting in the case of phenol leaving groups with pK(a) values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pK(a) values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl-enzyme, intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.
Place, publisher, year, edition, pages
2008. Vol. 47, no 29, 7762-7769 p.
end-specific cellobiohydrolase, transfer-catalyzed synthesis, cell-wall, polysaccharides, substrate-specificity, cellulomonas-fimi, cellulolytic, enzymes, structural-analysis, crystal-structures, degrading enzymes, expression
IdentifiersURN: urn:nbn:se:kth:diva-17693DOI: 10.1021/bi8009168ISI: 000257665100018ScopusID: 2-s2.0-47649125408OAI: oai:DiVA.org:kth-17693DiVA: diva2:335738
QC 201005252010-08-052010-08-05Bibliographically approved