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

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.

Automated summary

DXPS is essential in bacteria but absent in human metabolism, making it a potential antibacterial drug target; The enzyme possesses unique features that enable selective inhibition strategies and raise questions about its function in bacterial pathogens.