Kinetic analyses of retaining endo-(Xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides. Ibatullin, F., Baumann, M., Greffe, L., & Brumer, H. Biochemistry, 47(29):7762-7769, 2008. cited By 24
Kinetic analyses of retaining endo-(Xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides [link]Paper  doi  abstract   bibtex   
A library of phenyl β-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 Glc4 backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by β-galactosidase treament. Subsequent per-O-acetylation, α-bromination, phase-transfer glycosylation, and Zemplén deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pKa 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 pKa values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pKa 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. © 2008 American Chemical Society.
@ARTICLE{Ibatullin20087762,
author={Ibatullin, F.M. and Baumann, M.J. and Greffe, L. and Brumer, H.},
title={Kinetic analyses of retaining endo-(Xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides},
journal={Biochemistry},
year={2008},
volume={47},
number={29},
pages={7762-7769},
doi={10.1021/bi8009168},
note={cited By 24},
url={https://www.scopus.com/inward/record.uri?eid=2-s2.0-47649125408&doi=10.1021%2fbi8009168&partnerID=40&md5=66aea507deaaf913166a027f9f76526d},
affiliation={School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-106 91 Stockholm, Sweden; Petersburg Nuclear Physics Institute, Molecular and Radiation Biology Division, Russian Academy of Science, Gatchina, St. Petersburg 188300, Russian Federation},
abstract={A library of phenyl β-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 Glc4 backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by β-galactosidase treament. Subsequent per-O-acetylation, α-bromination, phase-transfer glycosylation, and Zemplén deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pKa 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 pKa values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pKa 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. © 2008 American Chemical Society.},
correspondence_address1={Brumer, H.; School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-106 91 Stockholm, Sweden; email: harry@biotech.kth.se},
issn={00062960},
coden={BICHA},
pubmed_id={18627132},
language={English},
abbrev_source_title={Biochemistry},
document_type={Article},
source={Scopus},
}

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