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 (αKG):UMP oxygenases that catalyze the formation of CO₂, 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′-²H]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 ²H atom of [1′,3′,4′,5′,5′-²H]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.