Enzymatic Assays to Investigate Acyl-Homoserine Lactone Autoinducer Synthases

Abstract

Bacteria use chemical molecules called autoinducers as votes to poll their numerical strength in a colony. This polling mechanism, commonly referred to as quorum sensing, enables bacteria to build a social network and provide a collective response for fighting off common threats. In Gram-negative bacteria, AHL synthases synthesize acyl-homoserine lactone (AHL) autoinducers to turn on the expression of several virulent genes including biofilm formation, protease secretion, and toxin production. Therefore, inhibiting AHL signal synthase would limit quorum sensing and virulence. In this chapter, we describe four enzymatic methods that could be adopted to investigate a broad array of AHL synthases. The enzymatic assays described here should accelerate our mechanistic understanding of quorum-sensing signal synthesis that could pave the way for discovery of potent antivirulence compounds.

In vitro synthesis and biochemical characterization of acyl-homoserine lactone synthase and its deletion mutant

“…In the presence of AHL synthase, nucleophilic attack of the SAM α-amino group to the C-1 carbonyl carbon of the acyl-ACP or acyl-CoA occurs (acylation), producing holo-ACP or HS-CoA from acyl-ACP or acyl-CoA, respectively [18]. Subsequently, a nucleophilic attack of α-carboxylate oxygen on the γ-carbon of SAM occurs, which is lactonization of SAM [18]. This results in the production of AHL and 5'-methyl-thioadenosine.…”

“…The protocol of the AHL synthase assay is applied from the general method using the DPIP redox reaction, with a specification to YpeI [18]. The protocol consists of three steps: determining background rates, determining optimal enzyme concentration, and specifying the enzymatic constant.…”