Mukherjee_2025_Biochemistry__

This study reveals dual catalytic activities (beta-lactamase and esterase) in a new penicillin-recognizing protein (IITRS), found in two closely related species, Enterococcus faecium and Enterococcus lactis. IITRS is distinct from other beta-lactamase classes, showing only limited structural and functional similarity to class C beta-lactamases. The conserved KTG motif, which helps in substrate recognition in class C, is not present in this enzyme. The enzyme is different from class C in terms of different conserved loops, such as R(2) and loops, which are involved in the recognition, specificity, and hydrolysis of beta-lactams. Nevertheless, the involvement of Ser64 and Tyr150 residues in beta-lactam hydrolysis as found in class C enzymes has been demonstrated by site-directed mutagenesis. The study also highlights Tyr150 from the catalytic triad Tyr-Asp-Lys as being responsible for the esterase activity. This dual functionality confers catalytic promiscuity, enabling IITRS to function through two different mechanisms. The enzyme exhibits hydrolysis of p-NP esters (acetate, butyrate, hexanoate, decanoate, and laurate) displaying progressively higher activity with increasing alkyl chain lengths. Since Tyr150 has been found as a common ligand-binding residue for both of the activities, the beta-lactamase inhibition by diisopropyl fluorophosphate (DFP), a reported inhibitor of bacterial esterase, also has been demonstrated. This promising albeit unexplored biocatalyst also might be used in the production of chiral compounds, investigating its enantioselective nature similar to other bacterial esterases. Overall, this research upholds a new promiscuous enzyme and proposes a distinct active site, narrower than that of a beta-lactamase and wider than that of an esterase.