title: Analysis of the functional contributions of Asn233 in metallo-β-lactamase IMP-1.
abstract: Metallo-β-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of β-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming β-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-β-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four β-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of β-lactams.
authors: Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
authors: Nicholas G
authors: NG
authors: Brown
authors: Lori B
authors: LB
authors: Horton
authors: Wanzhi
authors: W
authors: Huang
authors: Sompong
authors: S
authors: Vongpunsawad
authors: Timothy
authors: T
authors: Palzkill
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001216:Asparagine / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D001216:Asparagine; C409201:beta-lactamase IMP-1; D001618:beta-Lactamases