Analysis of Surface Binding Sites (SBS) within GH62, GH13, and GH77

Casper Wilkens, Darrell Cockburn, Susan Andersen, Bent Ole Petersen, Christian Ruzanski, Robert A. Field, Ole Hindsgaul, Hiroyuki Nakai, Barry McCleary, Alison M. Smith, Maher Abou Hachem, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    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 AnAbf62A-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 AnAbf62A-m2,3 model are proposed to form an SBS situated about 30 Å from the catalytic site. Compared to wild-type, W23A/Y44A AnAbf62A-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 AnAbf62A-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.
    Original languageEnglish
    JournalJournal of Applied Glycoscience
    Volume62
    Issue number3
    Pages (from-to)87-93
    Number of pages7
    ISSN1344-7882
    DOIs
    Publication statusPublished - 2015

    Keywords

    • Affinity gel electrophoresis
    • Surface plasmon resonance
    • Arabinofuranosidase
    • α-amylase
    • Arabinoxylan
    • Starch

    Cite this

    Wilkens, C., Cockburn, D., Andersen, S., Ole Petersen, B., Ruzanski, C., A. Field, R., Hindsgaul, O., Nakai, H., McCleary, B., M. Smith, A., Abou Hachem, M., & Svensson, B. (2015). Analysis of Surface Binding Sites (SBS) within GH62, GH13, and GH77. Journal of Applied Glycoscience, 62(3), 87-93. https://doi.org/10.5458/jag.jag.jag-2015_006