Plasmid-based class C beta-lactamases confer resistance to cephamycins (e.g., cefoxitin, cefotetan) due to their high hydrolytic rates (kcat &gt; 5 s-1). However, the active sites of chromosomal non-cephamycinases show few differences from those of plasmid-based cephamycinases. To determine the identities of the residues that contribute most to this increased rate of hydrolysis, a representative enzyme of each type (P99 cephalosporinase and FOX-4, respectively) was chosen for functional analysis. The genes, including their periplasmic localization sequences, were each cloned into an expression vector under a TEM-1 constitutive promoter. Then, several active site residues were mutagenized using site-saturation mutagenesis, generating libraries containing all 20 mutations at a single position. The libraries were transformed into Escherichia coli TG1 and screened for activity against a panel of cephalosporins and cephamycins to determine the minimum inhibitory concentration of the wild-type and variant enzymes. Variants showing the greatest effect on substrate selectivity toward cephamycins were overexpressed and purified. Michaelis-Menten kinetic analysis revealed the mechanistic role of each altered position.

Using site-saturation mutagenesis to identify residues that contribute to cephamycinase activity in class C beta-lactamases

253rd National Meeting (2017)

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