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标题：Analysis of Surface Binding Sites (SBS) within GH62, GH13, and GH77
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作者：Casper Wilkens ; Darrell Cockburn ; Susan Andersen 等
期刊名称：Journal of Applied Glycoscience
印刷版ISSN：1344-7882
电子版ISSN：1880-7291
出版年度：2015
卷号：62
期号：3
页码：87-93
DOI：10.5458/jag.jag.JAG-2015_006
出版社：The Japanese Society of Applied Glycoscience
摘要：Certain interactions between carbohydrate active enzymes and polysaccharides involve surface binding sites (SBS) situated on catalytic domains outside of the active site. We recently undertook to develop a toolbox for SBS identification and characterization. In affinity gel electrophoresis (AGE) SBS containing proteins are migrating slower in native polyacrylamide electrophoresis gels cast with polysaccharide versus without polysaccharide. Amylolytic enzymes from GH13 and GH77 and xylanases from GH10 and GH11 are the best studied GH families with respect to SBS, presenting about half of the reported SBSs. In GH13 SBSs have been seen in 17 subfamilies including SBSs with highly diverse functions in the same enzyme. Circumstantial evidence is provided for an SBS in the GH77 MalQ from Escherichia coli , the bacterial orthologue of Arabidopsis DPE2 involved in starch metabolism. Furthermore, Aspergillus nidulans α-L-arabinofuranosidase An Abf62A-m2,3 of GH62 that has very high activity on wheat arabinoxylan (WAX) shows an unusually strongly retarded migration by WAX during AGE analysis. Using a recent GH62 crystal structure as template, Trp²³ and Tyr⁴⁴ in an An Abf62A-m2,3 model are proposed to form an SBS situated about 30 Å from the catalytic site. Compared to wild-type, W23A/Y44A An Abf62A-m2,3 retained 45% activity on WAX and was less retarded in AGE by WAX as well as by barley β-glucan and birchwood xylan, which are neither hydrolysed nor inhibiting activity towards WAX. The presence of a functional SBS agrees with W23A/Y44A An Abf62A-m2,3 retaining only 3-25% activity for arabinoxylo-oligosaccharides (AXOS) of DP 3-5 possibly reflecting allosteric activation of wild-type through SBS occupation by AXOS.