Mechanism of Vibrio cholerae autoinducer-1 biosynthesis

Abstract

Vibrio cholerae, the causative agent of the disease cholera, uses a cell to cell communication process called quorum sensing to control biofilm formation and virulence factor production. The major V. cholerae quorum-sensing signal CAI-1 has been identified as (S)-3-hydroxytridecan-4-one, and the CqsA protein is required for CAI-1 production. However, the biosynthetic route to CAI-1 remains unclear. Here we report that (S)-adenosylmethionine (SAM) is one of the two biosynthetic substrates for CqsA. CqsA couples SAM and decanoyl-coenzyme A to produce a previously unknown but potent quorum-sensing molecule, 3-aminotridec-2-en-4-one (Ea-CAI-1). The CqsA mechanism is unique; it combines two enzymatic transformations, a β,γ-elimination of SAM and an acyltransferase reaction into a single PLP-dependent catalytic process. Ea-CAI-1 is subsequently converted to CAI-1, presumably through the intermediate tridecane-3,4-dione (DK-CAI-1). We propose that the Ea-CAI-1 to DK-CAI-1 conversion occurs spontaneously, and we identify the enzyme responsible for the subsequent step: conversion of DK-CAI-1 into CAI-1. SAM is the substrate for the synthesis of at least three different classes of quorum-sensing signal molecules, indicating that bacteria have evolved a strategy to leverage an abundant substrate for multiple signaling purposes.

Figure 1

Structures of CAI-1-type molecules. CAI-1, (S)-3-hydroxytridecan-4-one; Am-CAI-1, (S)-3-aminotridecan-4-one; Ea-CAI-1, 3-aminotridec-2-en-4-one; and DK-CAI-1, tridecane-3,4-dione are shown.

Figure 2

Potential substrates for CqsA and kinetic studies of SAM and LAB as CqsA substrates. (a) Different compounds were examined for activity as CqsA substrates. Reactions were carried out with each potential substrate at 1 mM and d-CoA at 100 μM. Control reactions with SAM only (no d-CoA), without CqsA, and with an inactive CqsA mutant CqsA K236A(13) are also shown. (b, c) Coenzyme A release was measured by a coupled enzyme assay. Kinetic constants for LAB (b) and SAM (c) were estimated by fitting initial velocities to the Michaelis−Menten equation using GraphPad Software.

Figure 3

Dose responses of the CqsS receptor for CAI-1 type molecules. V. cholerae CqsS dose responses are shown for CAI-1 (◆), Ea-CAI-1 (●), DK-CAI-1 (▲), and Am-CAI-1 (◼). Light production is shown at particular concentrations of compounds. Data were fit with a variable-slope sigmoidal dose−response curve.

Scheme 1. Proposed CqsA Reaction Scheme

In the CqsA reaction using SAM and d-CoA as substrates, we propose a reaction sequence that combines a β,γ-elimination of SAM with the release of MTA and an acyltransferase reaction with the release of CoA. V-Gly is likely an enzyme-bound intermediate (boxed). This scheme is based on this study and inspired by the schemes for ACCS and CqsA.^,, The lysine amino moiety in the figure depicts the active site Lys236 residue on CqsA.(13).

Figure 4

In vivo CAI-1 isotope labeling experiments. In vivo CAI-1 labeling experiments were performed by supplementing l-methionine or d₈-l-methionine in combination with glucose or ¹³C-glucose in M9 minimal medium. HRMS spectra are enlarged to show specific regions around molecular weights 215, 218, 225, and 228. Corresponding chemical structures, compound formulas, and expected molecular weights are indicated at the bottom of each column. In the structures, predicted deuterium atoms and ¹³C carbon atoms are denoted with “D” and “∗”, respectively.

Figure 5

Conversion of Ea-CAI-1/DK-CAI-1 to CAI-1. Conversion experiments were performed with a V. cholerae cqsA lysate (a, d) or lysis buffer (b, e) providing Ea-CAI-1 (a−c) or DK-CAI-1 (d−f) and excess NADPH. In a, b, d, and e, HRMS spectra are enlarged to show peaks for CAI-1 and for the internal standard, d₂-CAI-1, as noted. In c and f, dose responses measured by light production are shown for the CqsS* receptor for Ea-CAI-1 to CAI-1 conversion (c) and for the wild-type CqsS receptor for DK-CAI-1 to CAI-1 conversion (f). Products were diluted as noted for reactions with cqsA lysate (●) and buffer (◼).

Figure 6

Conversion experiments with VC1059-type enzymes and V. cholerae mutant lysates. (a) In vitro conversion of DK-CAI-1 to CAI-1 following provision of DK-CAI-1, NADPH, and one of the recombinant proteins: VCA0301 (◆), VCA0691 (◼), VC1591 (▲), VC2021 (○), and VC1059 (◻). NADPH decreases were monitored. At each time point, relative absorbance at 340 nm was calculated by subtracting the initial absorbance at time zero. The reading interval is shorter for VC1059 because of its rapid reaction speed. Only selected time points for VC1059 are shown after the absorbance reaches a plateau. Conversion of Ea-CAI-1 (b) or DK-CAI-1 (c) to CAI-1 was carried out with V. cholerae mutant lysates. Mutations are noted in the figure. Conversion efficiencies are normalized to that of the cqsA single mutant.

Scheme 2. CAI-1 Biosynthesis Scheme

SAM and d-CoA are the in vivo substrates for CqsA, which produces Ea-CAI-1, MTA, and CoASH. Ea-CAI-1 is converted to CAI-1 through the intermediate DK-CAI-1. We suggest that conversion of Ea-CAI-1 to DK-CAI-1 occurs spontaneously. VC1059 is the primary enzyme responsible for converting DK-CAI-1 to CAI-1.