OleB from Bacterial Hydrocarbon Biosynthesis Is a β‑Lactone Decarboxylase That Shares Key Features with Haloalkane Dehalogenases ChristensonJames K. RobinsonSerina L. EngelTiffany A. RichmanJack E. KimAn N. WackettLarry P. 2017 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 <i>Xanthomonas campestris</i> performs an unprecedented β-lactone decarboxylation reaction, to complete <i>cis</i>-olefin biosynthesis. OleB reactions monitored by <sup>1</sup>H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating <i>cis</i>-β-lactones and no discernible activity with <i>trans</i>-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 <i>oleABCD</i> gene clusters, suggesting a misannotation. OleB from <i>X. campestris</i> showed very low dehalogenase activity only against haloalkane substrates with long alkyl chains. A haloalkane substrate mimic alkylated wild-type <i>X. campestris</i> OleB but not OleB<sub>D114A</sub>, 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 <i>Micrococcus luteus</i> was demonstrated to have the same activity, stereochemical preference, and dependence on the proposed Asp nucleophile. H<sub>2</sub><sup>18</sup>O studies with <i>M. luteus</i> OleBC suggested that the canonical alkyl–enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as <sup>18</sup>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.