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

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, 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.

X-ray crystallography–based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate

“…In this study, we report the first crystal structures of DXPS in the presence of a pre-decarboxylation intermediate mimic, PLThDP, and the native post-decarboxylation enamine intermediate. These structures of DXPS from D. radiodurans were determined under anoxic conditions to prevent unwanted side reactions (24). Conformational rearrangements of the protein are observed near the active site in these bound states, which is consistent with previous HDX-MS results (25) and provide insights into the molecular basis by which protein conformational flexibility dictates LThDP reactivity.…”

“…Besides D-GAP, DXPS utilizes an array of triggers and acceptor substrates, such as O 2 , which induces LThDP decarboxylation in the absence of D-GAP and accepts two electrons from the C2␣carbanion to form peracetate (Fig. S1A) (22)(23)(24).…”

Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase

“…12 Ternary complex formation upon binding of D-GAP to the E-LThDP complex is required to facilitate LThDP decarboxylation (Figure 1, steps 3 and 4). [12][13][14] Characterization of DXP synthase oxygenase activity revealed a similar mechanism of LThDP decarboxylation induced by O 2 . Thus, D-GAP and O 2 are both triggers of LThDP decarboxylation on DXP synthase.…”

“…Previous kinetic analyses of H49 and H299 variants (and analogous variants on D. radiodurans DXP synthase) were conducted under aerobic conditions, 19,21 prior to the discovery that oxygenase activity interferes in mechanistic and structural studies of DXP synthase. 13,16 Here, we show that all H49 and H299 variants display significant, albeit inefficient, oxygenase activity ( Figure S1); accordingly, we conducted detailed kinetic analyses under anaerobic conditions, to exclude any potentially confounding effects of oxidative pyruvate decarboxylation in comparing kinetic parameters for H49A, H49N, H299A, H299N, and wild type DXP synthase. Variants were selected to determine the effects of both conservative (asparagine) and nonconservative (alanine) substitutions on substrate affinity, catalytic activity, and inhibition under anaerobic conditions.…”

“…Kinetic parameters of these variants are comparable under anaerobic and aerobic conditions, which is expected given the low catalytic efficiency of DXP synthase-catalyzed acetate formation compared to that of DXP formation (Table 1, Table S1, and Figures S2 and S3). 13 H49 DXP synthase variants display decreased catalytic efficiency with respect to both substrates in the presence or absence of oxygen, with substitution of either residue having more pronounced effects on k cat /K m pyruvate than on k cat /K m Figure S3). This points to a key role for H299 in the rate-limiting LThDP formation step and binding of pyruvatederived intermediates along the reaction coordinate.…”