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Protein Science (2001), 10:2556-2565.
Copyright © 2001 The Protein Society

Identification of residues critical for metallo-ß-lactamase function by codon randomization and selection

Isabel C. Materon1 and Timothy Palzkill1,2

1 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA
2 Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA

Reprint requests to: Dr. Timothy Palzkill, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; e-mail: timothyp{at}bcm.tmc.edu; fax: (713) 798-7375.

IMP-1 ß-lactamase is a zinc metallo-enzyme encoded by the transferable blaIMP-1 gene, which confers resistance to virtually all ß-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes ß-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the blaIMP-1 gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.

Keywords: ß-Lactam antibiotics; metallo-ß-lactamases; structure; function; antibiotic resistance; carbapenems; randomization mutagenesis


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