Environmental species from theTelluria group as the putative origin of bifunctionalβ-lactamases

β-Lactamases comprise two structurally and evolutionarily well-defined groups: serine- (SBL) and metallo-β-lactamases (MBL). To date, clinically relevant β-lactamases are typically monofunctional DD-peptidases, containing a single active site cavity per molecule. Recently, several genes encoding putative β-lactamases from the four molecular classes (named as LRA) were identified through functional metagenomics in Alaskan soil samples. bla_LRA-13 encoded a 609-amino acid protein encompassing a class D and a class C-like β-lactamase fused as a single polypeptide, translated from a single open reading frame (ORF). Furthermore, we identified 20 LRA-13 homologs, one of them found in a Duganella hordei isolate, sharing 91.3% amino acid identity. Predicted structures generated with AlphaFold 3 showed similar conserved architectures encompassing an N-terminal and C-terminal domains compatible with class D and class C β-lactamases, respectively, connected by a short peptide as a linker and containing their characteristic structural features. A maximum likelihood (ML) evolutionary tree showed a close relationship between LRA-13 and the putative β-lactamase from Duganella hordei, a species belonging to the Telluria group, indicating that bifunctional enzymes likely evolved from a common remote ancestor and that their diversification may provide an evolutionary advantage in certain environmental niches. The genetic content of bla_LRA-13 and related genes appears to have a conserved synteny. The description of β-lactamases with two catalytic sites constitutes a novel finding and provides a basis for exploring new evolutionary mechanisms.IMPORTANCEβ-Lactamases are enzymes able to destroy β-lactam antibiotics and are divided into two main groups according to their structural and mechanistic features: serine- (SBL) and metallo-β-lactamases (MBL). To date, β-lactamases that represent a threat and are produced by bacterial pathogens contain a unique catalytic "pocket,"i.e., only a single β-lactam molecule is bound and cleaved at a time. LRA-13 and other related proteins seem to contain two different catalytic sites of different kinds (one of them is related to class C β-lactamases and the other to class D enzymes). In this study, we analyzed if these enzymes can represent a different evolutionary path for the β-lactamases.