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Identification of residues critical for metallo‐β‐lactamase function by codon randomization and selection
Author(s) -
Materon Isabel C.,
Palzkill Timothy
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.40884
Subject(s) - amino acid , mutant , biology , biochemistry , escherichia coli , amp resistance , enzyme , residue (chemistry) , active site , alanine , gene , chemistry , stereochemistry
IMP‐1 β‐lactamase is a zinc metallo‐enzyme encoded by the transferable bla IMP‐1 gene, which confers resistance to virtually all β‐lactam antibiotics including carbapenems. To understand how IMP‐1 recognizes and hydrolyzes β‐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.