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 1H 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 OleBD114A, 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. H218O studies with M. luteus OleBC suggested that the canonical alkyl-enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as 18O 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.
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*E-mail: email@example.com. ORCID James K. Christenson: 0000-0002-2597-9951 Funding The authors acknowledge the support of the Biotechnology Institute of the University of Minnesota and the MnDRIVE Initiative from the Office of the Vice President for Research of the University of Minnesota (L.P.W.). S.L.R. was supported by a National Science Foundation Graduate Research Fellowship (Grant 00039202). Notes The authors declare no competing financial interest.