Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.
CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Zhizeng
Z
Sun
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Shrenik C
SC
Mehta
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Carolyn J
CJ
Adamski
Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Richard A
RA
Gibbs
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Timothy
T
Palzkill
D016980:Aeromonas hydrophila / Q000201:enzymology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015780:Carbapenems / Q000737:chemistry; D020134:Catalytic Domain; D017124:Conserved Sequence; D059014:High-Throughput Nucleotide Sequencing; D006868:Hydrolysis; D007700:Kinetics; D020125:Mutation, Missense; D013379:Substrate Specificity; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D015780:Carbapenems; D001618:beta-Lactamases; C082063:cphA protein, Aeromonas hydrophila