Functional dynamics of water in New Delhi metallo‐β‐lactamase catalysis

Water plays a critical role in the hydrolysis of antibiotics by New Delhi metallo-β-lactamases (NDMs). The reaction proceeds through a nucleophilic attack on the β-lactam ring, followed by cleavage of the C-N bond and formation of an anionic nitrogen intermediate. This intermediate is then protonated by a water molecule diffusing from the bulk solvent. Although the catalytic mechanism of NDMs has been extensively studied, the molecular mechanism governing water entry into the active site during hydrolysis remains poorly understood. Here, we use molecular dynamics simulations to characterize the dynamics of catalytic water molecules during NDM-mediated hydrolysis. We examine the NDM-1, substrate-bound, intermediate, and product states of the NDM-1-cefiderocol system. In the enzyme-intermediate state, conformational rearrangements of loops L3 and L10, together with cefiderocol coordination to Zn2, expand the active site and increase the Zn1-Zn2 separation to ~4.5 Å. These changes promote formation of a water channel toward Zn2, allowing water to enter the active site and protonate the anionic nitrogen, thereby completing hydrolysis. Concurrently, structural changes restrict the water-entry pathway associated with Zn1. These findings reveal how local protein dynamics and water influx couple to catalysis, providing mechanistic insights into NDM function and informing strategies to inhibit these enzymes.