Probing the Mechanism of LipL: A non-heme, Fe(II)-Dependent α-ketoglutarate:UMP Dioxygenase that Initiates the Biosynthesis of High Carbon Nucleoside Antibiotics

Several nucleoside natural product antibiotics from Streptomyces sp. and actinomycetes have recently been shown to target bacterial peptidoglycan cell wall biosynthesis by inhibiting the bacterial translocase I (MraY). The biosynthetic gene clusters for A-90289, liposidomycins and caprazamycins revealed a protein with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD). This enzyme (LipL) is a mononuclear, non-heme, Fe(II) dependent α-KG:UMP dioxygenase responsible for the net dephosphorylation and two electron oxidation of uridine monophosphate (UMP) to uridine-5'-aldehyde. The postulated radical mechanism involving the formation of an unstable hydroxylated intermediate is investigated via the characterization of a key product obtained from the reaction of LipL (and its homolog Cpr19) with a synthetically modified surrogate substrate where the bridging phosphoester oxygen in UMP is replaced with a 5' C-P bond. We also exploit the broad substrate specificity of Cpr19 as a means of conducting precursor-directing biosynthesis with a view to generating interesting novel intermediates in the biosynthetic pathway with potential improved therapeutic values. The development of ¹⁸O_2, H₂ ¹⁷O and elemental sulfur labeled UMP is also discussed as an alternate strategy to tracking the fate of the eliminated phosphate.