Hydration Changes are Essential for Efficient Breakdown of Faropenem by the L1 Metallo-β-lactamase

Abstract

Abstract Metallo-β-lactamases (MBLs) are key contributors to antimicrobial resistance (AMR). The L1 MBL, originally from Stenotrophomonas maltophilia, is a tetrameric, dizinc, subclass B3 MBL that efficiently hydrolyzes most β-lactam antibiotics and evades inhibitors that act on other MBLs. Using quantum mechanics/molecular mechanics (DFTB3/MM) simulations with enhanced sampling, we mapped the full reaction pathway and minimum free energy profile for L1-catalyzed hydrolysis of faropenem, a penem antibiotic whose reaction can yield alternative enamine and imine products. Benchmarking against B3LYP-D3/6-31G(d) shows that DFTB3 provides the best correlation between energy and structure for this system. We test different possible mechanisms and find that changes in hydration at the metal site during the catalytic cycle are critical for efficient hydrolysis. Our results show that a zinc-bridging hydroxide acts as the nucleophile, with the conserved active site residue D120 participating in back-and-forth proton-transfer events and stabilizing the enzyme-intermediate (EI) complex. The direct reaction of EI, however, involves a large energy barrier; the second stage is facilitated by diffusion of a water molecule into the active site. This water molecule was observed during long-timescale MM MD simulations using the Extended Zinc Amber Force Field (EZAFF). Direct proton transfer from this water to the substrate amide nitrogen forms an enamine product, while imine formation requires rotation of the faropenem 6α-hydroxyethyl substituent to enable proton transfer from water to the substrate C2. The calculated free energy barriers for the formation of both products are consistent with the experimentally determined catalytic constant, and the calculated greater stability of the imine with its crystallographic observation. These simulations provide dynamic insight into L1 catalysis, highlighting features that can be exploited for inhibitor design.

The authors' abstract, as published at the source. ACS Catalysis, 2026 · DOI ↗