title: Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-β-Lactamase Identified from a Functional Metagenomic Study.
abstract: β-Lactamase production increasingly threatens the effectiveness of β-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known β-lactamases and mobilization from environmental reservoirs. The spread of metallo-β-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other β-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant β-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure. blaRm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high Km values. An Rm3 crystal structure reveals the MBL superfamily αβ/βα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for β-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of β-lactam resistance.
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom.
authors.forename: Ramya
authors.initials: R
authors.lastname: Salimraj
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Lihong
authors.initials: L
authors.lastname: Zhang
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom.
authors.forename: Philip
authors.initials: P
authors.lastname: Hinchliffe
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Elizabeth M H
authors.initials: EM
authors.lastname: Wellington
authors.affiliation: Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brem
authors.affiliation: Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: William H
authors.initials: WH
authors.lastname: Gaze
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom Jim.Spencer@bristol.ac.uk.
authors.forename: James
authors.initials: J
authors.lastname: Spencer
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D056186:Metagenomics / Q000379:methods*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001618:beta-Lactamases