Disruption of an Active Site Network Leads to Activation of C2α-Lactylthiamin Diphosphate on the Antibacterial Target 1-Deoxy-d-xylulose-5-phosphate Synthase

Abstract

The bacterial metabolic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and Dglyceraldehyde-3-phosphate (D-GAP). DXP is an essential bacteriaspecific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of D-GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of D-GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote LThDP activation. Here, we show that the substitution of putative network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. Substitutions predicted to disrupt the network have the effect to activate LThDP for decarboxylation, resulting in CO 2 and acetate production. In contrast, a substitution predicted to strengthen the network fails to activate LThDP and has the effect to shift DXPS toward the closed state. Network-disrupting substitutions near the carboxylate of LThDP also have a pronounced effect to shift DXPS to an open state. These results offer initial insights to explain the long-lived LThDP intermediate and its activation through disruption of an active site network, which is unique to DXPS. These findings have important implications for DXPS function in bacteria and its development as an antibacterial target.

Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog

“…Our recent studies of Ec R99 (analogous to Dr K101) suggest that this residue functions in an active site network that coordinates LThDP on the long-lived E-LThDP complex in its closed state; thus, this network may also be important for stabilizing PLThDP adducts. Based on the proximity of K101 to the benzyl group of 1 (Figure ), we hypothesized that a cation−π interaction may also contribute to the potent DXPS inhibition observed for 1 .…”

“…To determine inhibitor potency, we used the DXPS-DXP reductoisomerae (DXPS-IspC) coupled assay to measure the activity of recombinant purified Ec DXPS ,,,, in the presence of inhibitors. Reactions were initiated by the addition of substrates following a short preincubation of the inhibitor with enzymes, and initial velocities were determined by monitoring reduced nicotinamide adenine dinucleotide phosphate (NADPH) absorbance at 340 nm.…”

“…We reasoned that 8 should also engage the pyruvate binding site and react with ThDP to form a PLThDP adduct. Circular dichroism (CD) is a powerful tool that can be used to detect LThDP and LThDP-like adducts on thiamin-dependent enzymes, ,− including DXPS. ,,,,− , DXPS bound to ThDP (E-ThDP) is characterized by a CD λ min at 320 nm, indicative of the 4′-aminopyrimidine (AP) tautomer of ThDP (Figure A). Upon addition of pyruvate to E-ThDP, a new positive signal emerges at 313 nm, indicating the formation of LThDP in its 1′,4′-iminopyrimidine (IP) form.…”

“…Antimicrobial resistance predates the discovery of antibiotics and is an unavoidable threat in the treatment of infectious diseases. To combat resistance, new antibacterial strategies are needed that are selective for pathogens and avoid toxicity to the host microbiome, which can negatively impact human health. − Agents that target central metabolic processes are increasingly sought − and have the potential to exert narrow-spectrum activities. ,− However, this target space is relatively underexploited owing to the complexities in developing central metabolism targets and the perceived difficulties in selectively targeting bacterial metabolism over host metabolism. ,,, Essential to bacterial central metabolism and absent in humans, 1-deoxy- d -xylulose 5-phosphate synthase (DXPS) emerged as a promising antibacterial target with the potential to offer opportunities for developing narrow spectrum approaches. ,− …”

“…Interest in the development of DXPS as an antibacterial target has motivated studies of DXPS homologues to identify targetable features of this enzyme. ,,,− In fact, DXPS has many attributes which make it a promising therapeutic target, including a large active site compared to mammalian ThDP-dependent enzymes, , remarkable substrate promiscuity, ,,, and a unique mechanism in ThDP enzymology. ,,,− Mechanistic studies of Escherichia coli DXPS elucidated a conformationally driven ligand-gated mechanism (Figure A), in which ternary complex formation (E-LThDP-GAP) is required for efficient decarboxylation of the first enzyme-bound intermediate in the reaction pathway, C2α-lactylThDP (E-LThDP). ,,,, Unique to the DXPS mechanism, LThDP is long-lived on DXPS in a closed conformation. ,, Binding of d -GAP causes a shift in DXPS conformation to an open form that coincides with LThDP decarboxylation. , The resulting carbanion (E-carbanion) reacts with d -GAP as an acceptor substrate in a carboligation event to form DXP. In addition to d -GAP, DXPS responds to other inducers of LThDP decarboxylation and acceptor substrates, including O 2 , which induces LThDP decarboxylation in the absence of the natural acceptor and is subsequently reduced by the C2α-carbanion en route to peracetate …”