Abstract
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, CO2 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. O2-dependent LThDP decarboxylation led to one-electron transfer from the C2α-carbanion/enamine to O2, with intermediate ThDP-enamine radical formation, followed by peracetic acid formation en route to acetate. Thus, LThDP formation and decarboxylation and DXP formation were studied under anaerobic conditions. Our results support a model in which O2-dependent LThDP decarboxylation and peracetic acid formation occur in the absence of d-GAP, decreasing the levels of pyruvate and O2 in solution. The relative pyruvate and O2 concentrations then dictate the extent of LThDP accumulation, and its buildup can be observed when [pyruvate] > [O2]. The finding that O2 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.
Highlights
Important aspects of the DXP synthase mechanism are uncovered, gaps in our understanding of catalysis on this unique ThDP-dependent enzyme remain
DXP formation is the only known physiological function of DXP synthase, and our results indicate the possibility that O2 may compete with D-GAP in the LThDP decarboxylation and/or carboligation steps leading to DXP formation
The rates of LThDP decarboxylation (k2) upon addition of O2-saturated buffer or deoxygenated buffer containing D-GAP to preformed LThDP were 62.0 Ϯ 8.9 and 50.3 Ϯ 7.8 sϪ1, respectively (Figs. 12, A–C, Table 3), comparable with the rate of LThDP decarboxylation in the presence of D-GAP under aerobic conditions previously reported (k2 ϭ 42 Ϯ 1 sϪ1) [15]. These results suggest that LThDP decarboxylation occurs efficiently in the presence of either D-GAP or O2
Summary
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). In contrast to other ThDP-dependent enzymes that utilize classical pingpong kinetics, DXP synthase stabilizes LThDP and requires ternary complex formation upon binding of D-GAP to trigger efficient decarboxylation of LThDP by an unknown mechanism [13, 15] These mechanistic insights have guided selective inhibitor design [5,6,7,8]. Circular dichroism (CD) and NMR studies subsequently revealed the accumulation of LThDP on DXP synthase in the absence of D-GAP, and provided evidence that D-GAP plays a role to induce efficient LThDP decarboxylation [15] These findings are consistent with a mechanism involving ternary complex formation on DXP synthase; these results do not explain the earlier observation that DXP synthase. This study provides key insights into our understanding of a DXP synthase mechanism and raises interesting questions about the function of the unique requirement for ternary complex formation on DXP synthase
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