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Impact of remote mutations on metallo-β-lactamase substrate specificity: implications for the evolution of antibiotic resistance

Oelschlaeger, Peter and Mayo, Stephen L. and Pleiss, Juergen (2005) Impact of remote mutations on metallo-β-lactamase substrate specificity: implications for the evolution of antibiotic resistance. Protein Science, 14 (3). pp. 765-774. ISSN 0961-8368. http://resolver.caltech.edu/CaltechAUTHORS:20110913-173427641

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Abstract

Metallo-β-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of β-lactam antibiotics. The G262S point mutation distinguishing the metallo-β-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.


Item Type:Article
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http://dx.doi.org/10.1110/ps.041093405 DOIUNSPECIFIED
http://onlinelibrary.wiley.com/doi/10.1110/ps.041093405/abstractPublisherUNSPECIFIED
Additional Information:© 2005 The Protein Society. Published by Cold Spring Harbor Laboratory Press. Received September 1, 2004; Final revision October 19, 2004; Accepted November 11, 2004. Article first published online: 1 Jan 2009. We thank Jeffrey H. Toney (formerly with Merck) for providing a plasmid containing the IMP-1 gene, Merck for the gift of imipenem, and Marie Ary for assistance with the manuscript. This work was supported by the Colvin Biology Fellowship at Caltech (to P.O.), the Howard Hughes Medical Institute, the Ralph M. Parsons Foundation, and an IBM Shared University research grant (to S.L.M.).
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Funding AgencyGrant Number
Colvin Biology FellowshipUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
IBM Shared University Research GrantUNSPECIFIED
Subject Keywords:Biological Evolution; Drug Resistance: Bacterial; Escherichia coli; Mutation; Substrate Specificity; Drug Design; Kinetics; beta-Lactamases
Record Number:CaltechAUTHORS:20110913-173427641
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20110913-173427641
Official Citation:Oelschlaeger, P., Mayo, S. L. and Pleiss, J. (2005), Impact of remote mutations on metallo-β-lactamase substrate specificity: Implications for the evolution of antibiotic resistance. Protein Science, 14: 765–774. doi: 10.1110/ps.041093405
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25338
Collection:CaltechAUTHORS
Deposited By: Marie Ary
Deposited On:23 Sep 2011 21:40
Last Modified:23 Sep 2011 21:40

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