Structures of the Michaelis complex (1.2 Å) and the covalent acyl intermediate (2.0 Å) of cefamandole bound in the active sites of the Mycobacterium tuberculosis β-lactamase K73A and E166A mutants

Biochemistry. 2010 Nov 16;49(45):9685-7. doi: 10.1021/bi1015088. Epub 2010 Oct 25.

Abstract

The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active β-lactamase, BlaC, that imparts TB with resistance to β-lactam chemotherapy. The structure of covalent BlaC-β-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 Å, respectively. These structures provide insight into the details of the catalytic mechanism.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Catalytic Domain
  • Cefamandole / metabolism*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Genome, Bacterial
  • Kinetics
  • Models, Molecular
  • Mycobacterium tuberculosis / enzymology
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism*
  • Protein Binding
  • beta-Lactamases / chemistry
  • beta-Lactamases / genetics*
  • beta-Lactamases / metabolism*

Substances

  • DNA, Bacterial
  • Cefamandole
  • beta-Lactamases

Associated data

  • PDB/3N8S
  • PDB/3NY4