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Hydroxyl groups in the ββ sandwich of metallo-β-lactamases favor enzyme activity: a computational protein design study

Oelschlaeger, Peter and Mayo, Stephen L. (2005) Hydroxyl groups in the ββ sandwich of metallo-β-lactamases favor enzyme activity: a computational protein design study. Journal of Molecular Biology, 350 (3). pp. 395-401. ISSN 0022-2836. http://resolver.caltech.edu/CaltechAUTHORS:20110620-160427419

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Abstract

Metallo-β-lactamases challenge antimicrobial therapies by their ability to hydrolyze and inactivate a broad spectrum of β-lactam antibiotics. The potential of these enzymes to acquire enhanced catalytic efficiency through mutation is of great concern. Here, we explore the potential of computational protein design to predict mutants of the imipenemase IMP-1 that modulate the catalytic efficiency of the enzyme against a range of substrates. Focusing on the four amino acid positions 69, 121, 218, and 262, we carried out a number of design calculations. Two mutant enzymes were predicted: the single mutant S262A and the double mutant F218Y-S262A. Compared to IMP-1, the single mutant (S262A) results in the loss of a hydroxyl group and the double mutant (F218Y-S262A) results in a hydroxyl transfer from position 262 to position 218. The presence of both hydroxyl groups at positions 218 and 262 was tested by examining the mutant F218Y. Kinetic constants of IMP-1, the two computationally designed mutants (S262A and F218Y-S262A), and the hydroxyl addition mutant (F218Y) were determined with seven substrates. Catalytic efficiencies are highest for the enzyme with both hydroxyl groups (F218Y) and lowest for the enzyme lacking both hydroxyl groups (S262A). The catalytic efficiencies of the two enzymes with one hydroxyl group each are intermediate, with the F218Y-S262A double mutant exhibiting enhanced hydrolysis of nitrocefin, cephalothin, and cefotaxime relative to IMP-1.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.jmb.2005.04.044 DOIUNSPECIFIED
http://www.sciencedirect.com/science/article/pii/S0022283605004651PublisherUNSPECIFIED
Additional Information:© 2005 Elsevier Ltd. Received 17 February 2005; Accepted 21 April 2005. Edited by J. Thornton. Available online 11 May 2005. 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 Howard Hughes Medical Institute, DARPA, the Ralph M. Parsons Foundation, an IBM Shared University Research grant (to S.L.M.) and the Caltech Colvin Fellowship (to P.O.).
Funders:
Funding AgencyGrant Number
Howard Hughes Medical InstituteUNSPECIFIED
Ralph M. Parsons Foundation UNSPECIFIED
IBM Shared University Research GrantUNSPECIFIED
Caltech Colvin FellowshipUNSPECIFIED
Defense Advanced Research Projects Agency (DARPA)UNSPECIFIED
Subject Keywords:Binding Sites; beta-Lactamases; Bacteroides fragilis; Models: Molecular; Proteins; Protein Structure: Secondary; Circular Dichroism; Protein Folding; Point Mutation; Computational Biology; Software; Substrate Specificity; Catalysis; Kinetics; Zinc; Temperature; Proteomics; metallo-β-lactamase; zinc β-lactamase; protein design; catalytic efficiency
Record Number:CaltechAUTHORS:20110620-160427419
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20110620-160427419
Official Citation:Peter Oelschlaeger, Stephen L. Mayo, Hydroxyl Groups in the ββ Sandwich of Metallo-β-lactamases Favor Enzyme Activity: A Computational Protein Design Study, Journal of Molecular Biology, Volume 350, Issue 3, 15 July 2005, Pages 395-401, ISSN 0022-2836, 10.1016/j.jmb.2005.04.044.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:24088
Collection:CaltechAUTHORS
Deposited By: Marie Ary
Deposited On:28 Sep 2011 22:02
Last Modified:28 Sep 2011 22:02

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