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Defining the role of a unique Ω‐loop insertion in the class D β‐lactamase OXA‐1
Author(s) -
Bopra Angela M.,
Leonard David A.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1017
Subject(s) - cefepime , enzyme kinetics , cefotaxime , substrate (aquarium) , chemistry , cephalosporin , mutagenesis , enzyme , stereochemistry , penicillin , loop (graph theory) , active site , biology , antibiotics , biochemistry , mutation , mathematics , antibiotic resistance , gene , imipenem , combinatorics , ecology
In Class A, C and D β‐lactamases, an Ω‐loop is found near the active site and is involved in both catalysis and substrate selection. Among the class D oxacillinases, OXA‐1 has the largest Ω‐loop, which is six hydrophilic residues longer than in other OXA enzymes. To determine the effect of the extended Ω‐loop found in OXA‐1 on substrate affinity, we removed the six additional residues using polymerase chain reaction mutagenesis. Minimum inhibitory concentration (MIC) testing showed that the deletion provided no significant alteration in resistance to most penicillin and cephalosporin antibiotics; one main exception to this was a four‐fold enhancement of the MIC for cefepime. The enzyme was then purified and the protein was subjected to kinetic analysis. For cefotaxime, the K m for the OXA‐1 Ω‐loop deletion showed no large difference and the K cat was slightly lower than wild‐type. However significant differences were found for cefepime compared to wild‐type OXA‐1. The K m and the K cat values were significantly higher, suggesting that the deletion of the Ω‐loop has an effect both on binding and catalysis for cefepime.