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CAU-1, a Subclass B3 Metallo-ß-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in the Caulobacter crescentus Chromosome

Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, 53100 Siena,¹ Dipartimento di Sanità Pubblica, Sezione di Microbiologia, Università di Roma "La Sapienza," 00185 Rome,² Dipartimento di Biologia, Università di Roma "Tor Vergata," 00133 Rome,³ Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, 67100 L'Aquila, Italy,⁴ Laboratoire d'Enzymologie et Centre d'Ingénierie des Protéines, Institut de Chimie, Université de Liège, Sart Tilman, B-4000 Liège, Belgium,⁵ RW Johnson Pharmaceutical Research Institute, Raritan, New Jersey⁶

Received 15 October 2001/ Returned for modification 3 January 2002/ Accepted 10 March 2002

The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-ß-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-ß-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was 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 of Stenotrophomonas 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 ß-lactams were poor overall (K_m values 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. In C. crescentus, the CAU-1 enzyme is produced independently of ß-lactam exposure and, interestingly, the bla_CAU determinant 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-ß-lactamase in a member of the subdivision of the class Proteobacteria.

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