Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Abstract

The thiamin diphosphate-dependent enzyme 1-deoxy- d -xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d -glyceraldehyde 3-phosphate ( d -GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d -GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor–acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction

“…This DHEThDP carbanion/enamine formed from HPA or glycolaldehyde on DXPS is the same physiological intermediate formed on TK, raising a question about whether DXPS processes this intermediate in a manner similar to TK. Interestingly, DXPS avoids dehydration of the DHEThDP carbanion to form the dead-end acetyl-ThDP . This behavior is similar to TK and is distinct from most other ThDP enzymes, hinting at the possibility that this could be a physiologically relevant intermediate on DXPS (Figure ).…”

“…coli DXP synthase and E. coli IspC were overexpressed and purified as previously reported. ,,, A vinyl anaerobic chamber from Coy Laboratory Products (Grass Lake, MI, USA) was used for all anaerobic experiments. Two Tecan Infinite M Nano U/V visible plate readers and a BioTek Epoch 2 microplate reader were used: one Tecan and the BioTek under ambient conditions for aerobic spectrophotometric analyses and the other Tecan situated inside the Coy chamber for anaerobic spectrophotometric analyses.…”

“…O 2 consumption was detected using an oxygen electrode as described previously. , Unless otherwise noted, reactions were performed in the presence of 5 μM DXPS and 1 mM substrate in buffer containing 100 mM of HEPES buffer at pH 8, 100 mM of NaCl, 2 mM of MgCl 2 , and 1 mM of ThDP. The electrode was calibrated daily at 25 °C, and solutions were prepared in the absence of substrate and equilibrated at 25 °C for a minimum of 2 min until the O 2 signal stabilized.…”

“…Further supporting this notion, we have shown that DXPS displays both substrate and catalytic promiscuity with reactivities that may be driven by conformational dynamics. DXPS utilizes several ketoacid and aldehyde donor substrates, , and d -GAP, glyceraldehyde, or molecular oxygen act as small molecule triggers of decarboxylation. − A variety of acceptor substrates are also utilized by DXPS in the presence of pyruvate, including aliphatic/aromatic aldehydes and ketoacids in C–C formation, , aryl nitroso compounds in C–N formation, and even O 2 in C–O formation . The catalytic promiscuity of DXPS is evidenced by its oxygenase activity in which pyruvate is converted to peracetate via the ThDP-enamine radical and superoxide formed by electron transfer from the carbanion to O 2 .…”

The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway

“…Some thiaminrequiring enzymes may also be susceptible to damage from sidereactions involving reactive intermediates. For example, in addition to its primary activity of catalyzing synthesis of DXS from pyruvate and glyceraldehyde-3-P, bacterial DXS has an oxygenase sideactivity that results in decarboxylation of pyruvate forming a reactive peracetate intermediate in the active site (Johnston and Meyers, 2021). Conceivably, if the oxygenase side-reaction occurs in chloroplasts where O 2 concentrations are high, this reaction could be a significant cause of damage to the enzyme, shortening its functional lifetime.…”