Dual activity of PNGM-1, a metallo-β-lactamase and tRNase Z, pinpoints the evolutionary origin of subclass B3 metallo-β-lactamases

Antibiotic resistance is a steadily increasing global problem which could lead to a fundamental upheaval in clinical care with the potential to return us to the pre-antibiotic era 1-4 . The production of β-lactamases, a group of enzymes that confer antibiotic resistance in Gram-negative bacteria, is now one of the major barriers in treating Gram-negative infections 5 . β-Lactamases are classified according to their catalytic mechanisms into serine β-lactamases and metallo-β-lactamases 6,7 . There are functional and structural similarities between serine β-lactamases and penicillin-binding proteins, and so serine β-lactamases are thought to have evolved from a penicillin-binding protein 7,8 . Given the functional and structural differences between serine β-lactamases and metallo-β-lactamases, metallo-β-lactamases are thought to have evolved from a protein other than a penicillin-binding protein, but to date this ancestor remains unknown 8-11 . We discovered PNGM-1, the first subclass B3 metallo-β-lactamase, in deep-sea sediments that predate the antibiotic era 12 . Here we discover the dual activity of PNGM-1, pinpointing the evolutionary origin of subclass B3 metallo-β-lactamases. Phylogenetic analysis suggested that PNGM-1 could yield insights into the evolutionary origin of subclass B3 metallo-β-lactamases. We reveal the structural similarities between tRNase Zs and PNGM-1, which prompted us to investigate their evolutionary relationship and the possibility of them possessing dual enzymatic activities. We demonstrate that PNGM-1 has dual activity with both true metallo-β-lactamase and tRNase Z activity, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 metallo-β-lactamases and tRNase Zs. These comparisons revealed that the B3 metallo-β-lactamase activity of PNGM-1 is a promiscuous activity and subclass B3 metallo-β-lactamases are thought to have evolved through PNGM-1 activity. Our work provides a foundation for the evolution of tRNase Z into subclass B3 metallo-β-lactamases through the dual activity of PNGM-1.