期刊
PROTEIN SCIENCE
卷 23, 期 10, 页码 1451-1460出版社
WILEY
DOI: 10.1002/pro.2530
关键词
metallo-beta-lactamase; point mutation; antibiotic resistance; enzyme evolution; IMP-1 antibody
资金
- NCATS NIH HHS [UL1 TR001425] Funding Source: Medline
- NIAID NIH HHS [R01 AI100560, R01 AI063517] Funding Source: Medline
In Gram-negative bacteria, resistance to beta-lactam antibacterials is largely due to beta-lactamases and is a growing public health threat. One of the most concerning beta-lactamases to evolve in bacteria are the Class B enzymes, the metallo-beta-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel beta-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of beta-lactam antibacterials. Catalytic efficiencies (k(cat)/K-M) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in beta-lactam turnover and support this approach to predict activities of certain novel MBL variants.
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