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The N152T mutation in the class C beta‐lactamase, AmpC, can serve as a substrate selectivity switch
Author(s) -
Baggett Vincent,
Docter Brianne,
Powers Rachel,
Wallar Brad
Publication year - 2013
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.27.1_supplement.561.15
Subject(s) - active site , asparagine , selectivity , enterobacter cloacae , residue (chemistry) , mutant , chemistry , substrate (aquarium) , stereochemistry , saturated mutagenesis , mutation , enzyme , biochemistry , biology , escherichia coli , enterobacteriaceae , gene , ecology , catalysis
The class C β‐lactamase, AmpC, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. While some active site residues have been characterized to be involved in the recognition and breakdown of the substrate, many of their specific roles in substrate selectivity are not fully understood. Previous work on the Enterobacter cloacae P99 cephalosporinase demonstrated that a mutation of a highly conserved residue in the active site (N152) can result in substrate selectivity changes. Here, we investigate the role of the active site residue asparagine‐152 (N152) in E. coli AmpC by mutating it to a threonine (T) residue and examining the effect that this mutation has on kinetic and structural properties with different β‐lactam drugs. We discovered that although the N152T mutation caused higher K m values with all substrates, N152T exhibits over 150 fold higher k cat value against cefotaxime. In addition, the N152T mutation provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC. To probe the mechanism behind the observed substrate selectivity switch, the crystal structure of AmpC N125T was determined at 2.05 Å. In comparison to the wild type AmpC structure, the small structural differences in the active site have been associated with the changes in the K m and k cat kinetic values as a way to uncover the specific role of N152 in the function of AmpC.