Cyclobutanone Inhibitor of Cobalt-Functionalized Metallo-γ-Lactonase AiiA with Cyclobutanone Ring Opening in the Active Site

Abstract

An α-amido cyclobutanone possessing a C10 hydrocarbon tail was designed as a potential transition-state mimetic for the quorum-quenching metallo-γ-lactonase autoinducer inactivator A (AiiA) with the support of in-house modeling techniques and found to be a competitive inhibitor of dicobalt­(II) AiiA with an inhibition constant of K i = 0.007 ± 0.002 mM. The catalytic mechanism of AiiA was further explored using our product-based transition-state modeling (PBTSM) computational approach, providing substrate-intermediate models arising during enzyme turnover and further insight into substrate–enzyme interactions governing native substrate catalysis. These interactions were targeted in the docking of cyclobutanone hydrates into the active site of AiiA. The X-ray crystal structure of dicobalt­(II) AiiA cocrystallized with this cyclobutanone inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)­decanamide ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl chain and its interaction with the hydrophobic phenylalanine clamp region of AiiA adjacent to the active site enabled atomic placement of the ligand atoms, including the C10 alkyl chain. A mechanistic hypothesis for the ring opening is proposed involving a radical-mediated process.

Quorum‐quenching enzymes: Promising bioresources and their opportunities and challenges as alternative bacteriostatic agents in food industry

“…Therefore, the structural analysis of phosphotriesterase-like lactonase SsoBox showed that the Trp263 on the active site loop is crucial for regulating the substrate specificity. A recent study established substrateintermediate models existing in the process of enzyme turnover using product-based transition-state modeling to support the hypothesis of catalytic mechanism involving the radical-mediated process (Reidl et al, 2021). Although the substrate catalytic site is consistent for multiple AHL lactonases, some studies have reported α/β hydrolase family AHL lactonases AidH and AiiM that do not have (Kaihao & Xiao-Hua, 2014) (1), predicted active center (Nain et al, 2020) (2), and catalytic mechanism (Bokhove et al, 2010;Liu et al, 2008) (3) of AHL lactonase (a) and ahl acylases (b) characteristic metal-binding active site motifs HXHXDH, suggesting there may be differences in the detailed catalytic mechanisms (Gao et al, 2013).…”