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The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) β-lactamase from Aeromonas hydrophila is a Zn2+-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His118, Asp120, His196 and His263) and in substrate specificity (Val67, Thr157, Lys224 and Lys226), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp120 and His263, and thus is located in the ‘cysteine’ zinc-binding site. His118 and His196 residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val67 plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val67 also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys224 in the binding of substrate. Lys226 is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding. [ABSTRACT FROM AUTHOR]

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl–enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase. [ABSTRACT FROM AUTHOR]