LAUSR

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.