Mechanism of a Standalone β-Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme.
Enzyme-catalyzed β-lactone formation from β-hydroxy acids is a crucial step in bacterial biosynthesis of β-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β-lactone synthetase activity using synthetic β-hydroxy acid substrates with alkene or alkyne moieties. β-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain β-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the β-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the β-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.
['Robinson|Serina L|SL|0000-0001-6947-7913', 'Christenson|James K|JK|0000-0002-2597-9951', 'Richman|Jack E|JE|', 'Jenkins|Dominick J|DJ|', 'Neres|João|J|0000-0003-4488-2423', 'Fonseca|Dallas R|DR|', 'Aldrich|Courtney C|CC|0000-0001-9261-594X', 'Wackett|Lawrence P|LP|']
D000255:Adenosine Triphosphate D000595:Amino Acid Sequence D019757:Carbon-Oxygen Lyases D002384:Catalysis D020134:Catalytic Domain D057075:Enzyme Assays D006880:Hydroxy Acids D007700:Kinetics D008274:Magnesium D008956:Models, Chemical D016297:Mutagenesis, Site-Directed D010802:Phylogeny D011485:Protein Binding D016415:Sequence Alignment D013379:Substrate Specificity D016959:Xanthomonas campestris
ANL superfamily biosynthesis natural products adenylation beta-lactone synthetase