CAU-1, a subclass B3 metallo-beta-lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome

The sequenced chromosome ofCaulobacter crescentusCB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned inEscherichia colifromC. crescentustype strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced inE. coliwas purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme ofStenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Kmvalues were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. InC. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, theblaCAUdeterminant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the classProteobacteria.