Mechanism of a Standalone β‐Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme

Abstract

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.

Global analysis of adenylate-forming enzymes reveals β-lactone biosynthesis pathway in pathogenic Nocardia

“…S6B). These results are consistent with the reported adenylation activity for the β-lactone synthetase from Xanthomonas campestris and with most enzymes in the ANL superfamily (30). Overall, these results support AdenylPred-guided predictions that NltC in N. brasiliensis is a functional β-lactone synthetase.…”

“…The formation of the unstable β-keto acid catalyzed by OleA and NltAB could be observed as its ketone breakdown product by GC-MS as published previously (33). Olefins from the complete thermal decarboxylation of β-lactone products were detected without derivatization by comparison to synthetic β-lactone standards described previously (8,30). The β-hydroxy acids required methylation of the carboxylic acid group by diazomethane for detection by GC-MS. Ethereal alcoholic solutions of diazomethane were prepared from N-methyl-Nnitroso-p-toluenesulfonamide (Sigma-Aldrich).…”

“…Within the ANL superfamily, the β-lactone synthetases were the most recently discovered family and the extent of their role in natural product biosynthesis remains poorly understood (8,30). We used AdenylPred to identify >90 β-lactone synthetases in uncharacterized biosynthetic gene clusters from 48 different genera (Fig.…”

Spirolactone, an unprecedented antifungalβ-lactone spiroketal macrolide fromStreptomyces iranensis

“…Currently, there are only few enzymatic mechanisms corresponding to β -lactone ring formation that have been reported, 47 including: (1) the intramolecular cyclization from seven-membered ring, catalyzed by cyclase VibC in vibralactone biosynthesis; 48 (2) the tandem aldol-lactonization bicyclization reaction to generate the γ -lactam- β -lactone structure, catalyzed by standalone ketosynthase SalC in salinosporamide A biosynthesis; 49 (3) the β -lactone formation during the intramolecular attack of the β -hydroxyl group onto the thioester carbonyl, catalyzed by the C-terminal TE domain of ObiF in obafluorin biosynthesis or esterase GloD in globilactone A biosynthesis; 50,51 (4) the conversion of β -hydroxyl to β -lactone, catalyzed by β -lactone synthase OleC in olefin biosynthesis. 52,53 We proposed that the beta-lactone formation in spirolactone involves a two-step oxidation to form a carboxylic acid, followed by dehydration. It is likely that either the cytochrome P450 enzyme SplK or SplL is involved in the process, and we also identified several genes with unknown functions, such as splJ and splI .…”