Jyoti Barman, Partho Biswas, Partha Roy, Saugata Hazra
Abstract The widespread use and misuse of β-lactam antibiotics exert selective pressure on bacteria, driving the continuous emergence of novel metallo-β-lactamases (MBLs). Characterizing newly evolved MBLs is critical for elucidating the molecular basis of their antimicrobial resistance (AMR) and may contribute to designing novel inhibitors. In this study, we have done the functional characterization of an MBL-fold metallo-hydrolase from Klebsiella pneumoniae (designated Kmh-1), which shares low sequence similarity with B1 MBLs. Unlike B1 enzymes, where zinc binding motifs are strictly conserved (H116–H118–H196 and D120–C221–H263), Kmh-1 retains only Asp120 and His263, marking its clear divergence. Phylogenetic analysis further establishes its divergence from the existing B1 MBLs. Kmh-1 has activity toward diverse β-lactams, and zinc is required for enzymatic activity. This study also finds aztreonam hydrolyzing activity in this enzyme. Kmh-1–aztreonam interaction is validated through circular dichroism (CD) and fluorescence spectroscopy. AlphaFold structure prediction and docking studies confirm that Kmh-1 can accommodate aztreonam in the active site and disclose the probable aztreonam binding residues. Our results demonstrate a plausible explanation of effective aztreonam binding in Kmh-1, which is similar to that of clinically relevant NDM-1. IC50 values against different inhibitors are determined for Kmh-1, and the IC50 value against bicyclic boronate is observed in the nM range. Together, these findings establish Kmh-1 as an MBL-fold metallo-hydrolase endowed with aztreonam binding and hydrolysis, expanding the landscape of resistance and providing a framework for inhibitor design.