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

Abstract

IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes beta-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 bla(IMP-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.

Fig. 1.

Summary of sequencing results for the naïve IMP-1 random libraries. An average of 9 random mutants were sequenced from each naïve library. The amino acids found among the random mutants are shown below the labeled amino acid position. Each labeled amino acid position represents a distinct library. Stop codons and frameshift mutations are represented by an asterisk. The numbers in superscript represent the number of occurrences of the mutant among sequenced clones.

Fig. 2.

Summary of sequencing results of mutants selected from the IMP-1 random libraries based on their ability to confer ampicillin resistance to Escherichia coli. The amino acids found among the functional mutants are shown above the labeled amino acid position. Each labeled amino acid position represents a distinct library. The numbers in superscript represent the number of times the mutant appeared in the selection.

Fig. 3.

Spacefill diagram of the IMP-1 metallo-β-lactamase structure showing the mutagenized residues of the 27 random libraries. The residues are divided into three groups: critical, represents positions that do not allow any substitutions (red); restrictive, represents positions that allow 1–2 substitutions (yellow); and tolerant, represents positions that allow 3–6 substitutions (blue). The two green spheres represent zinc molecules. The figure was rendered using the Molscript and Raster3d programs (Kraulis 1991; Merritt and Murphy 1994) using coordinates from the Protein Data Bank accession code 1DD6 (Concha et al. 2000).