The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

Abstract

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release > or =100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.

Figure 1. Structural and functional analysis of CqsA

(a) The x-ray structure (model B; see Methods) of CqsA, with monomers depicted in cyan and magenta. The two PLP molecules are shown as space-filling models. (b) Stereo-view of the proposed PLP-amino-CAI-1 product aldimine modeled into the large cavity observed within the CqsA homodimer. The product aldimine is shown as a stick representation, with oxygen red, phosphorus orange, nitrogen blue, and carbon atoms yellow (PLP) and gray (amino-CAI-1). The cavity boundary, calculated using the program VOIDOO, is shown as a purple mesh. (c) Stereo-view of the side chains (green) contacting the modeled product aldimine in panel (b) (except for those side chains in contact with PLP). Residue labels are color-coded according to the strength of defect caused by substitution of each residue with alanine (Table S2), with red labels for those residues whose substitution resulted in <0.05% residual autoinducer production.

Figure 2. Activity of in vitro synthesized amino-CAI-1 and related compounds

(a) Purified CqsA (20 μM) was incubated with 10 mM SAB and 100 μM dCoA and the evolution of amino-CAI-1 was measured using mass spectrometry (filled squares, left Y-axis) and the V. cholerae bioluminescence assay (open squares, right Y-axis). No product is detected by mass spectrometry (filled symbols) or the bioluminescence assay (open symbols) when either SAB (triangles) or dCoA (circles) is omitted, or when catalytically inactive CqsA (K236A, diamonds) is substituted for the wild-type enzyme. Relative light units (RLU) are defined as counts min⁻¹ml⁻¹/OD₆₀₀. (b) Activity of synthetic (S)- and (R)-amino-CAI-1 (filled and open triangles, respectively) and (S)- and (R)-CAI-1 (filled and open squares, respectively) in the V. cholerae bioluminescence reporter assay.

Figure 4

Scheme 1. CAI-1, AON, and amino-CAI-1

(a) CAI-1; (S)-3-hydroxytridecan-4-one. (b) The AONS reaction. AONS catalyzes the production of the α-aminoketone (S)-8-amino-7-oxononanoate (AON) from L-alanine and pimeloyl-CoA. (c) The proposed CqsA reaction, catalyzing the production of the α-aminoketone amino-CAI-1 from SAB ((S)-2-aminobutyrate) and dCoA (decanoyl-coenzyme A).