title: In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone.
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 Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll 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 Rhll-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 Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.
authors.affiliation: Department of Pharmaceutical Sciences, University of Nottingham, UK.
authors.forename: Y
authors.initials: Y
authors.lastname: Jiang
authors.forename: M
authors.initials: M
authors.lastname: Camara
authors.forename: S R
authors.initials: SR
authors.lastname: Chhabra
authors.forename: K R
authors.initials: KR
authors.lastname: Hardie
authors.forename: B W
authors.initials: BW
authors.lastname: Bycroft
authors.forename: A
authors.initials: A
authors.lastname: Lazdunski
authors.forename: G P
authors.initials: GP
authors.lastname: Salmond
authors.forename: G S
authors.initials: GS
authors.lastname: Stewart
authors.forename: P
authors.initials: P
authors.lastname: Williams
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002569:Cerulenin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007700:Kinetics; D008025:Ligases; D013058:Mass Spectrometry; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C092312:N-butyrylhomoserine lactone; C104908:N-hexanoyl-L-homoserine lactone; D011994:Recombinant Proteins; D014157:Transcription Factors; C088386:homoserine lactone; D002569:Cerulenin; D000105:Acetyl Coenzyme A; D012436:S-Adenosylmethionine; D008025:Ligases; C096955:RHLI protein, Pseudomonas aeruginosa; D015107:4-Butyrolactone