title: 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.
abstract: 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.
authors.affiliation: Protein Engineering Network Centres of Excellence of Canada and the Department of Chemistry, University of British Columbia, Vancouver, V6T 1Z1 Canada.
authors.forename: Spencer J
authors.initials: SJ
authors.lastname: Williams
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: Michael N G
authors.initials: MN
authors.lastname: James
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010080:Oxazoles / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry*
chemical_list: D001386:Azides; D007477:Ions; D010080:Oxazoles; D001224:Aspartic Acid; D006821:Hyaluronoglucosaminidase; D001619:beta-N-Acetylhexosaminidases