Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria.

The underexploited antibacterial target 1-deoxy-d-xyluose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP). DXP is an essential intermediate in the biosynthesis of ThDP, pyridoxal phosphate, and isoprenoids in many pathogenic bacteria. DXP synthase catalyzes a distinct mechanism in ThDP decarboxylative enzymology in which the first enzyme-bound pre-decarboxylation intermediate, C2α-lactyl-ThDP (LThDP), is stabilized by DXP synthase in the absence of d-GAP, and d-GAP then induces efficient LThDP decarboxylation. Despite the observed LThDP accumulation and lack of evidence for C2α-carbanion formation in the absence of d-GAP, CO is released at appreciable levels under these conditions. Here, seeking to resolve these conflicting observations, we show that DXP synthase catalyzes the oxidative decarboxylation of pyruvate under conditions in which LThDP accumulates. O-dependent LThDP decarboxylation led to one-electron transfer from the C2α-carbanion/enamine to O, with intermediate ThDP-enamine radical formation, followed by peracetic acid formation to acetate. Thus, LThDP formation and decarboxylation and DXP formation were studied under anaerobic conditions. Our results support a model in which O-dependent LThDP decarboxylation and peracetic acid formation occur in the absence of d-GAP, decreasing the levels of pyruvate and O in solution. The relative pyruvate and O concentrations then dictate the extent of LThDP accumulation, and its buildup can be observed when [pyruvate] > [O]. The finding that O acts as a structurally distinct trigger of LThDP decarboxylation supports the hypothesis that a mechanism involving small molecule-dependent LThDP decarboxylation equips DXP synthase for diverse, yet uncharacterized cellular functions.