Functional analysis of the active site of a metallo-beta-lactamase proliferating in Japan

An R-plasmid-mediated metallo-beta-lactamase was found in Klebsiella pneumo niae DK4 isolated in Japan in 1991, The nucleotide sequence of its structur al gene revealed that the beta-lactamase termed DK4 was identical to the IM P-1 metallo-beta-lactamase which was mediated by a chromosomal gene of Serr atia marcescens TN9106 isolated in Japan in 1991 (E, Osano et al,, Antimicr ob. Agents Chemother. 38:71-78, 1994). The dose effect of DK4 beta-lactamas e production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the beta-lactams except monoba ctams. The enzymatic characteristics of the DK4 beta-lactamase and its kine tic parameters for nine beta-lactams were examined. The DK4 beta-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoen zyme that lacked the two zincs was structurally unstable, and the activitie s of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indic ated that His86, His88, and His149 sen ed as ligands to one of the zincs an d that Cys168 played a role as a ligand to the second zinc. Both zinc molec ules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid a t position 90 was replaced by asparagine, This mutant enzyme showed an appr oximately 1,000 times lower k(cat) value for cephalothin than that of the w ild-type enzyme but retained the two zincs even after dialysis against zinc -free buffer. The observed effect of pH on the activity suggested that Asp9 0 functions as a general base in the enzymatic process.