Molecular basis for the substrate specificity of quorum signal synthases

Abstract

In several Proteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl-homoserine lactones (AHL) signals using S-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 S-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.

Keywords: crystallography; homoserine lactone; quorum sensing.

Fig. 1.

The AHL synthase reaction mechanism. (A) Overall reaction scheme showing the BjaI-catalyzed production of isovaleryl–AHL from isovaleryl–CoA and S-adenosylmethionine via a presumptive isovaleryl–SAM intermediate. (B) Formation of isovaleryl–AHL analyzed by HPLC analysis (green trace). The elution profiles for the isolated standards are shown in the panels above. (C) Michaelis–Menten curve obtained by measuring CoA production over varying concentrations of isovaleryl–CoA (IV-CoA), at a fixed concentration of 300 μM S-adenosylmethionine. The slight decrease in initial rate may be due to substrate inhibition, which has previously been observed for ACP-dependent AHL synthases (31).

Fig. 2.

Substrate scope of BjaI. (A) Structures of various branched acyl–CoA produced by semisynthesis. (B) End-point liquid-chromatography–mass spectrometric analysis showing the production of acyl–homoserine lactone (in blue) from the corresponding acyl–CoA (in red) using BjaI. Experiments were conducted in triplicate, and error bars represent the SD between measurements.

Fig. 3.

Cocrystal structures of BjaI. (A) Ribbon diagram of BjaI in complex with methyl thioadenosine (MTA, in pink) and isovaleryl–CoA (IV-CoA, in green). Secondary structure elements are demarcated, and helix α1, which is mobile in the absence of nucleotide, is colored in deep red. (B) Active-site pocket showing the two cavities that accommodated substrates SAM (in pink) and acyl–CoA (isopentyl–CoA, in green). Trp34 (in yellow) is located adjacent to both cavities. (C–F) Simulated annealing difference Fourier maps (Fo-Fc) of BjaI complexes contoured to 2.5σ (blue) showing the bound ligands and important active-site residues. Protein residues are shown in gray, MTA/SAM is shown in pink, and the acyl–CoA is shown in green.

Fig. 4.

Observation of the acyl–SAH intermediate. (A) HPLC analysis of the reaction time course demonstrating the production of a covalent isovaleryl–SAH intermediate (elution time of 15 min) from SAH (elution time near 5 min). The identity of all products was confirmed by mass spectrometry. (B) Simulated annealing difference Fourier maps (Fo-Fc) of BjaI complexes contoured to 2.5σ (blue) showing the bound isovaleryl–SAH intermediate (in pink) and active-site residues (in gray). (C) Superposition of the BjaI complex structures with isovaleryl–SAH (in pink) and SAH (in green). Note the change in the orientation of the carboxylate, which would facilitate lactone formation. (D and E) Proposed mechanisms for the breakdown of the acyl–SAH intermediate to form the lactone product via either a (D) concerted S_N2-like mechanism or (E) distributive E2-type mechanism. The former mechanism occurs through a more favorable orbital overlap.

Fig. 5.

Structure-based classification of AHL synthases. (A and B) Sequence similarity network illustrating the relationship among different AHL synthases. (A) Alignment cutoff of at least 45% yielded 18 different groups, which clustered almost entirely based on organismal class, while (B) a more stringent cutoff of at least 50% yields the network that is clustered based on predicted substrate preference. The CoA-dependent AHL synthases can be further subdivided into four clades based on the CoA-linked acyl donor. (C) Divergence in sequence among the different CoA-dependent clade sequences near the “indole platform.” (D) Michaelis–Menten curve obtained by measuring CoA production by MesI over varying concentrations of either octanoyl–CoA (black curve) or octanoyl–ACP (red curve), at a fixed concentration of 1 mM S-adenosylmethionine.