A Comparative Analysis of Acyl‐Homoserine Lactone Synthase Assays

Abstract

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.

Enzymatic Assays to Investigate Acyl-Homoserine Lactone Autoinducer Synthases

“…5 Unfortunately, most AHL synthases still remain uncharacterized, perhaps due to challenges associated with studying this class of enzymes such as: i) the substrates for AHL synthase have low turnover (k cat ) and high nonenzymatic background rates, which affects the accuracy in the determination of true initial rates ii) neither the substrates nor the products absorb in a unique region in the electromagnetic spectrum necessitating the need for a reporter system to measure enzymatic rates iii) signal synthases are slow enzymes that do not appear to be selected for catalytic efficiency. 6 As a result, determination of enzymatic rates for mutant enzymes/substrate analogs becomes non-trivial. Assays developed for signal synthases must therefore be robust and versatile for characterizing wide array of AHL synthases.…”

Acyl-Homoserine Lactone Based Modulators for RhlI, a Quorum Sensing Signal Synthase in Pseudomonas aeruginosa

“…DCPIP colorimetric assay is a well-established assay used to determine AHL synthase activity by monitoring the enzyme-dependent rate of release of holo-ACP/CoA thiol product over time (Figure 7). [51][52][53] DCPIP, in its oxidized form, is a blue compound that absorbs at 600 nm (ε600 = 21,000 M -1 cm -1 = 2.1 x 10 -2 µM -1 cm -1 ). The holo-ACP thiol released upon acylation of SAM reduces the DCPIP dye to a colorless form, DCPIPH2.…”

“…As reported previously, non-specific reduction of DCPIP is a major limitation of the DCPIP colorimetric assay, which is compounded by significant levels of contamination in commercially available SAM-Cl. 53 To circumvent the issue, the reaction mixture, except the enzyme, was incubated with DCPIP and the decrease in absorbance at 600 nm was observed. The rate of decrease of absorbance flattens around 600 s (10 min) and the background rate in the 600-900 s range is equivalent of 0.013 µM/min, about 5% of the lowest enzyme rate observed in this project (Figure 26).…”

Investigation of Catalytic Activity of Stable β-Ketoacyl-ACP Substrate Analogs in Quorum Sensing Signal Synthesis

“…A coupled assay is designed to follow the release of MTA from lactonization reaction of acyl-ACP and SAM indirectly by coupling it to methylthioadenosine nucleosidase-xanthine oxidase (MTAN-XO) reactions (Scheme 3). 36 The concentrations of nucleosidase and xanthine oxidase are made to keep the AHL-synthase reaction ratelimiting. In this assay, the MTA product from AHL-synthase reaction first reacts with MTAN to form adenine and methylthioribose (MTR).…”

“…Two separate methods were designed for separating protein-based substrates form small molecule substrates. 36 The HPLC method used to monitor acylation half-reaction detects protein-based analytes (holo-ACP, acyl-ACP), while the HPLC method for monitoring lactonization half-reaction resolves small molecules (MTA, SAM). In lactonization assay, SAM is the reactant, and MTA is the product.…”