In several , LuxI-type enzymes catalyze the biosynthesis of acyl-homoserine lactones (AHL) signals using-adenosyl-l-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA-aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoA-dependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure-function, and kinetic analysis define the features that facilitate the unusual acyl conjugation with -adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight how a prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling.
Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their host by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-L-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has shown to be a difficult road. Two assays including a radiolabel and a colorimetric (DCPIP) assay are well documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage, and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA and acyl-ACP utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.
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