In vitro biosynthesis of the Pseudomonas aeruginosa quorum‐sensing signal molecule N‐butanoyl‐L‐homoserine lactone

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Abstract
In Pseudomonas aeruginosa, synthesis of the quorum‐sensing signal molecules N‐butanoyl‐L‐homoserine lactone (BHL) and N‐hexanoyl‐L‐homoserine lactone (HHL) requires the LuxI homologue RhlI(VsmI). By using thin‐layer chromatography in conjunction with high‐performance liquid chromatography (HPLC) and mass spectrometry, we show that purified RhlI can catalyse the biosynthesis of BHL and HHL using either S‐adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for RhlI‐directed N‐acylhomoserine lactone (AHL) biosynthesis. The N‐acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n‐butanoyl‐CoA or n‐hexanoyl‐CoA). The specificity of RhlI for charged CoA derivatives is demonstrated as RhlI was unable to generate AHLs detectable in our bioassays from acetyl‐CoA, malonyl‐CoA, n‐octanoyl‐CoA, n‐decanoyl‐CoA, DL‐β‐hydroxybutanoyl‐CoA or crotonoyl‐CoA. RhlI was also unable to use N‐acetyl‐S‐3‐oxobutanoylcysteamine, a chemical mimic for 3‐oxobutanoyl‐CoA. Furthermore, the RhlI‐catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.