Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas ) β-lactamase from Aeromonas hydrophila is a Zn 2+ -containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His 118 , Asp 120 , His 196 and His 263 ) and in substrate specificity (Val 67 , Thr 157 , Lys 224 and Lys 226 ), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp 120 and His 263 , and thus is located in the ‘cysteine’ zinc-binding site. His 118 and His 196 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. Val 67 plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val 67 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 Lys 224 in the binding of substrate. Lys 226 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.