Evidence that oxidative dephosphorylation by the nonheme Fe( II ), α‐ketoglutarate: UMP oxygenase occurs by stereospecific hydroxylation

LipL and Cpr19 are nonheme, mononuclear Fe( II )‐dependent, α‐ketoglutarate (α KG ): UMP oxygenases that catalyze the formation of CO 2 , succinate, phosphate, and uridine‐5′‐aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes—[1′,3′,4′,5′,5′‐ 2 H] UMP and the phosphonate derivative of UMP —and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2 H atom of [1′,3′,4′,5′,5′‐ 2 H] UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5′ R )‐5′‐hydroxy‐ UMP . Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase‐phospholyases.