PDBsum entry: 1m01

Hydrolase

PDB id: 1m01

Contents

Protein chain

499 a.a. *

Ligands

NAG

SO4

GOL ×3

Metals

_CL ×3

Waters ×280

* Residue conservation analysis

PDB id:

1m01

Name:

Hydrolase

Title:

Wildtype streptomyces plicatus beta-hexosaminidase in comple product (glcnac)

Structure:

Beta-n-acetylhexosaminidase. Chain: a. Synonym: beta-hexosaminidase, sphex. Engineered: yes

Source:

Streptomyces plicatus. Organism_taxid: 1922. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PQS)

Resolution:

2.10Å R-factor: 0.195 R-free: 0.216

Authors:

S.J Williams,B.L.Mark,D.J.Vocadlo,M.N.G.James,S.G.Withers

Key ref:

S.J.Williams et al. (2002). 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. PubMed id: 12171933 DOI: 10.1074/jbc.M206481200

Date:

11-Jun-02 Release date: 21-Jan-03

Protein chain

O85361  (O85361_STRPL) -  B-N-acetylhexosaminidase

Seq: 506 a.a.

Struc: 499 a.a.

 Gene Ontology (GO) functional annotation 

• Biological process: carbohydrate metabolic process (1 term)
• Biochemical function: catalytic activity (4 terms)

DOI no: 10.1074/jbc.M206481200

J Biol Chem 277:40055-40065 (2002)

PubMed id: 12171933

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.

S.J.Williams, B.L.Mark, D.J.Vocadlo, M.N.James, S.G.Withers.

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.

Selected figure(s)

Figure 9.
Fig. 9. Proposed mechanism of azide-mediated chemical rescue with the SpHex D313A variant. Azide as an alternative nucleophile to water acts to open the oxazolinium ion intermediate.

Figure 10.
Fig. 10. Stereographic superposition of NAG bound in the active sites of wild-type SpHex (gray), SpHex_ D313A (green), and SpHex_D313N (yellow). Of the two NAG conformations refined in the SpHex_D313A·NAG complex, only the catalytically incompetent conformer is shown. Panels A and B are oriented ~90° about the y-axis with respect to each other.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 40055-40065) copyright 2002.

Figures were selected by an automated process.