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-lactamase substrate specificity: Implications for the evolution of antibiotic resistance
1 Division of Biology and
2 Howard Hughes Medical Institute, Divisions of Biology and Chemistry/Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
3 Institute of Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany
(RECEIVED September 1, 2004; FINAL REVISION October 19, 2004; ACCEPTED November 11, 2004)
Metallo-
-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of -lactam antibiotics. The G262S point mutation distinguishing the metallo--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.
Keywords: metallo--lactamase; metalloenzyme; substrate specificity; enzyme evolution; point mutation
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041093405.
Reprint requests to: Juergen Pleiss, Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany; e-mail: Juergen.Pleiss{at}itb.uni-stuttgart.de; fax: +49-711-685-3196.
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