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

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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.