Structural and Biochemical Characterization of Rm3, a SubClass B3 Metallo- β-Lactamase Identified from a Functional Metagenomic Study

β-Lactamase production increasingly threatens the effectiveness of β-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge both through mutation of previously known β-lactamases and mobilization from environmental reservoirs. The spread of metallo-β-lactamases (MBLs) represents a particular challenge through their typically broad spectrum activities, encompassing carbapenems in addition to other β-lactam classes. Increasingly, genomic and metagenomic studies reveal 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, remains uncharacterized. Here we characterize the product of one such gene, bla_Rm3, identified through functional metagenomic sampling of an environment with high biocide exposure. bla_Rm3 encodes a subclass B3 MBL that, when expressed in recombinant E. coli, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high K_M values. An Rm3 crystal structure reveals the MBL superfamily αβ/βα fold, which more closely resembles mobilized B3 MBLs (AIM-1, 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 β-lactam affinity. 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 as an environmental reservoir of β-lactam resistance.