Expanding AHL Acylases Horizon - Insights From Structural Analysis of Choloylglycine Hydrolases FromShewanella lohicaPV-4

Abstract

Acyl homoserine lactone acylases are quorum quenching enzymes that degrade the Gram negative bacterial autoinducer N-acyl homoserine lactone (AHL) and belong to the Ntnhydrolases superfamily of enzymes. Recent findings reported AHL acylase activity of pencillin V acylases (PVA) which, alongside bile salt hydrolases, are members of the cholyolglycine hydrolase (CGH) family of the Ntn-hydrolases superfamily. The present study reports the unique activity profile of two CGHs from a marine bacterium Shewanella loihica-PV4, designated here as SlCGH1 and SlCGH2, including the structural analysis of SlCGH1. Both the enzymes exhibit AHL acylase activity while unexpectedly being inactive on standard CGH substrates PenV and bile salts. SlCGH1 differs from known homotetrameric CGHs in being a homodimer displaying a reduced active site volume attributed to loop orientation, which subsequently directs the substrate specificity. Moreover a ligand bound complex structure revealed an unusual bent conformation of the saturated acyl chain bound to the active site and also predicts a single oxyanion hole forming residue during catalysis instead of the usual two residues. Phylogenetic analysis reveals SlCGH1 homologs cluster separate from reported CGHs and AHL acylases. On the whole, SlCGH1 could represent a functionally distinct new sub-class of CGH as an adaptation to the marine environment and its structure could provide the structural framework for understanding such a novel subclass. We also make a modest proposal of a probable evolutionary link between AHL acylases and β lactam acylases based on the overlap in activity and structural features. Substrate promiscuity or cross-reactivity among different members of an enzyme superfamily can be useful tool to decipher functional and evolutionary relationships (1). The quorum quenching acylases acting on N-acylhomoserine lactones (AHLs) belong to the Ntn hydrolase superfamily, together withβ−lactam acylases such as penicillin G acylases (PGA), Penicillin V acylases (PVA) and cephalosporin acylases (CA). Recent findings (2-5) reveal structural similarity and a significant overlap in the substrate spectra of these enzymes, providing insights intoprobable links to bacterial signaling and antibiotic resistance (6).AHLs, produced by Gram-negative bacteria, are one of the most extensively studied signal moleculesfor bacterial communication or quorum-sensing (QS, 7,8). AHLacylases (catalyse amide bond hydrolysis) and lactonases (catalyse lactone ring hydrolysis) are the two major classes of enzymes that degrade AHLs, thus involved in quorum quenching (9-11).While AHL lactonases belong to three different enzyme superfamilies (12), a majority of AHL acylases belong to the Ntn hydrolase superfamily.Canonical AHL acylases, as Ntn hydrolases, are characterized by the presence of the αββα fold in their tertiary structure housing the N-terminal catalytic serine nucleophile, and cleave the amide bond between lactone ring and the fatty acyl side chain in AHLs (13). AHL acylases are closely related t...

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

“…AHL acylase is another typical QQ enzyme that cleaves AHLs at amide linkage between the acyl chain and homoserine lactone (Figure 3b). Since the first discovery of AiiD in V. paradoxus in 2000 (Leadbetter & Greenberg, 2000), additional AHL acylases such as Quip, PvdQ, HacA/B, Aca, AhlM, MacQ, and AhaP have been found in Pseudomonas , Acidovorax , Bacillus , Ralstonia , Shewanella , and Psychrobacter (Chen et al., 2013; Kaihao & Xiao‐Hua, 2014; Kusada et al., 2017; Philem et al., 2019; Reina et al., 2021). Based on the sequence analysis, AHL acylases formed two major clusters including QuiP cluster and AAC cluster (Figure 3b[1]).…”