Christenson_2017_Biochemistry_56_5278

OleB is an alpha/beta-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 beta-lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions monitored by (1)H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating cis-beta-lactones and no discernible activity with trans-beta-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. H2(18)O studies with M. luteus OleBC suggested that the canonical alkyl-enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as (18)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 beta-lactone decarboxylases, and posits an enzymatic mechanism of beta-lactone decarboxylation.