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

2001; Wiley; Volume: 10; Issue: 12 Linguagem: Inglês

10.1110/ps.ps.40884

1469-896X

Isabel C. Materon,

Machine Learning in Bioinformatics

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