1m01
Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)
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OverviewOverview
SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating, evidence suggests that the hydrolytic mechanism involves, substrate-assisted catalysis wherein the 2-acetamido substituent acts as a, nucleophile to form an oxazolinium ion intermediate. The role of a, conserved aspartate residue (D313) in the active site of SpHex was, investigated through kinetic and structural analyses of two variant, enzymes, D313A and D313N. Three-dimensional structures of the wild-type, and variant enzymes in product complexes with N-acetyl-d-glucosamine, revealed substantial differences. In the D313A variant the 2-acetamido, group was found in two conformations of which only one is able to aid in, catalysis through anchimeric assistance. The mutation D313N results in a, steric clash in the active site between Asn-313 and the 2-acetamido group, preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the, insensitivity of this variant to chemical rescue. By comparison, the D313A, mutation results in a shift in a shift in the pH optimum and a modest, decrease in activity that can be rescued by using azide as an exogenous, nucleophile. These structural and kinetic data provide evidence that, Asp-313 stabilizes the transition states flanking the oxazoline, intermediate and also assists to correctly orient the 2-acetamido group, for catalysis. Based on analogous conserved residues in the family 18, chitinases and family 56 hyaluronidases, the roles played by the Asp-313, residue is likely general for all hexosaminidases using a mechanism, involving substrate-assisted catalysis.
About this StructureAbout this Structure
1M01 is a Single protein structure of sequence from Streptomyces plicatus with NAG, CL, SO4 and GOL as ligands. Active as Beta-N-acetylhexosaminidase, with EC number 3.2.1.52 Full crystallographic information is available from OCA.
ReferenceReference
Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state., Williams SJ, Mark BL, Vocadlo DJ, James MN, Withers SG, J Biol Chem. 2002 Oct 18;277(42):40055-65. Epub 2002 Aug 8. PMID:12171933
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