This review focuses on biosynthesis of β-lactone rings in natural products. Biosynthetic routes to β-lactones and β-lactams are compared in the context of their chemical diversity and production by divergent organisms around the tree of life.

The first β-lactone synthetase enzyme is reported, creating an unexpected link between the biosynthesis of olefinic hydrocarbons and highly functionalized natural products. The enzyme OleC, involved in the microbial biosynthesis of long-chain olefinic hydrocarbons, reacts with syn- and anti-β-hydroxy acid substrates to yield cis- and trans-β-lactones, respectively. Protein sequence comparisons reveal that enzymes homologous to OleC are encoded in natural product gene clusters that generate β-lactone rings, suggesting a common mechanism of biosynthesis.

OleB is an α/β-hydrolase found in bacteria that biosynthesize long-chain olefinic hydrocarbons, but its function has remained obscure. We report that OleB from the Gram-negative bacterium Xanthomonas campestris performs an unprecedented β-lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions monitored by H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating cis-β-lactones and no discernible activity with trans-β-lactones, consistent with the known configuration of pathway intermediates. Protein sequence analyses showed OleB proteins were most related to haloalkane dehalogenases (HLDs) and retained the canonical Asp-His-Asp catalytic triad of HLDs. Unexpectedly, it was determined that an understudied subfamily, denoted as HLD-III, is comprised mostly of OleB proteins encoded within oleABCD gene clusters, suggesting a misannotation. OleB from X. campestris showed very low dehalogenase activity only against haloalkane substrates with long alkyl chains. A haloalkane substrate mimic alkylated wild-type X. campestris OleB but not OleB, implicating this residue as the active site nucleophile as in HLDs. A sequence-divergent OleB, found as part of a natural OleBC fusion and classified as an HLD-III, from the Gram-positive bacterium Micrococcus luteus was demonstrated to have the same activity, stereochemical preference, and dependence on the proposed Asp nucleophile. HO studies with M. luteus OleBC suggested that the canonical alkyl-enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as O is not incorporated into the nucleophilic aspartic acid. This work defines a previously unrecognized reaction in nature, functionally identifies some HLD-III enzymes as β-lactone decarboxylases, and posits an enzymatic mechanism of β-lactone decarboxylation.

Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His 6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive ␤-lactone intermediate produced by the OleC-catalyzed reaction.IMPORTANCE Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that longchain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in X. campestris. The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encode the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modelling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-coenzyme A substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is impacted by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer. Summary Statement OleA is the first reported thiolase enzyme whose active site general base, a glutamic acid (Glu117), is derived from a homodimeric interface. Structural and functional characterization of glutamatic acid mutants establishes its mechanistic role in initiating olefin biosynthesis.

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.

Renewable production of hydrocarbons is being pursued as a petroleum-independent source of commodity chemicals and replacement for biofuels. The bacterial biosynthesis of long-chain olefins represents one such platform. The process is initiated by OleA catalyzing the condensation of two fatty acyl-coenzyme A substrates to form a β-keto acid. Here, the mechanistic role of the conserved His285 is investigated through mutagenesis, activity assays, and X-ray crystallography. Our data demonstrate that His285 is required for product formation, influences the thiolase nucleophile Cys143 and the acyl-enzyme intermediate before and after transesterification, and orchestrates substrate coordination as a defining component of an oxyanion hole. As a consequence, His285 plays a key role in enabling a mechanistic strategy in OleA that is distinct from other thiolases.

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