Title | Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations. |
Publication Type | Journal Article |
Year of Publication | 2016 |
Authors | Sun, Z, Mehta, SC, Adamski, CJ, Gibbs, RA, Palzkill, T |
Journal | Sci Rep |
Volume | 6 |
Pagination | 33195 |
Date Published | 2016 Sep 12 |
ISSN | 2045-2322 |
Keywords | Aeromonas hydrophila, Amino Acid Sequence, Bacterial Proteins, Base Sequence, beta-Lactam Resistance, beta-Lactamases, Carbapenems, Catalytic Domain, Conserved Sequence, High-Throughput Nucleotide Sequencing, Hydrolysis, Kinetics, Mutation, Missense, Substrate Specificity |
Abstract | 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. |
DOI | 10.1038/srep33195 |
Alternate Journal | Sci Rep |
PubMed ID | 27616327 |
PubMed Central ID | PMC5018959 |
Grant List | R01 AI106863 / AI / NIAID NIH HHS / United States T32 GM008280 / GM / NIGMS NIH HHS / United States |
Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.
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