Functional diversity among metallo-beta-lactamases: characterization of the CAR-1 enzyme of Erwinia carotovora.
Metallo-beta-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available beta-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit 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 open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of Erwinia carotovora, an important plant pathogen. Although no detectable beta-lactamase activity could be found in E. carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several beta-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 lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. 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.
Stoczko
Magdalena
M
Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100, Siena, Italy.
Frère
Jean-Marie
JM
Rossolini
Gian Maria
GM
Docquier
Jean-Denis
JD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016973:Pectobacterium carotovorum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010641:Phenotype; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001618:beta-Lactamases