1M01

Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)

Summary for 1M01

• Entry DOI: 10.2210/pdb1m01/pdb
• Related: 1M03 1M04 1hp5 1jak
• Descriptor: Beta-N-acetylhexosaminidase, 2-acetamido-2-deoxy-beta-D-glucopyranose, CHLORIDE ION, ... (6 entities in total)
• Functional Keywords: substrate assisted catalysis, streptomyces plicatus, hexosaminidase, tim barrel, hydrolase
• Biological source: Streptomyces plicatus
• Total number of polymer chains: 1
• Total formula weight: 56826.69

Authors

J Williams, S.,Mark, B.L.,Vocadlo, D.J.,James, M.N.G.,Withers, S.G. (deposition date: 2002-06-11, release date: 2003-01-21, Last modification date: 2024-11-06)

Primary citation

Williams, S.J.,Mark, B.L.,Vocadlo, D.J.,James, M.N.,Withers, S.G.
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.
J.Biol.Chem., 277:40055-40065, 2002

PubMed 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.

PubMed: 12171933
DOI: 10.1074/jbc.M206481200

Experimental method

X-RAY DIFFRACTION (2.1 Å)