Functional Diversity among Metallo--lactamases: Characterization of the CAR-1 Enzyme of Erwinia carotovora

Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100, Siena, Italy; and Centre d'Ingénierie des Protéines & Laboratoire d'Enzymologie, Université de Liège, B-4000 Liège, Belgium

^* To whom correspondence should be addressed. Email: jddocquier{at}unisi.it.

	Abstract

Metallo--lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and their lack of sensitivity to commercially available -lactamase inactivators. Apart from the acquired subclass B1 enzymes that exhibit an increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an ORF (ECA2849) encoding a MBL-like protein in the sequenced genome Erwinia carotovora, an important plant pathogen. Although no detectable -lactamase activity could be found in Erwinia carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed a decreased susceptibility to several -lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime and cefotaxime and, to a less extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized by the enzyme. CAR-1 is the first example of a functional naturally-occurring MBL in the family Enterobacteriaceae (order Enterobacteriales) and highlights the extraordinary structural and functional diversity exhibited by MBLs.