Lactate monooxygenase (LMO) catalyzes the FMN‐dependent “coupled” oxidation of lactate and O2to acetate, carbon dioxide, and water, involving pyruvate and hydrogen peroxide as enzyme‐bound intermediates. Other α‐hydroxy acid oxidase family members follow an “uncoupled pathway,” wherein the α‐keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures of
This first crystal structure of the FMN‐dependent α‐hydroxy acid oxidase family member lactate monooxygenase (LMO) reveals it has a uniquely large active site lid that we hypothesize is stable enough to explain the slow dissociation of pyruvate that leads to its “coupled” oxidation of lactate and O2to produce acetate, carbon dioxide, and water. Also, the relatively widespread distribution of putative LMOs supports their importance and provides new motivation for their further study.