Differential Hydroxypyruvate Processing by <i>E. coli</i> and <i>P. aeruginosa</i> DXP Synthases Reveals Preferential Xylulose 5-Phosphate Formation by the <i>P. aeruginosa</i> Enzyme

The thiamine diphosphate (ThDP)-dependent enzyme 1-deoxy- d -xylulose 5-phosphate synthase (DXPS) converts pyruvate and d -glyceraldehyde 3-phosphate ( d -GAP) to the essential branchpoint metabolite, DXP. DXPS exhibits a ligand-gated mechanism where the predecarboxylation intermediate derived from pyruvate persists until d -GAP triggers decarboxylation. Together with its substrate/catalytic promiscuity and other features of multifunctionality, ligand gating on DXPS may poise this enzyme to catalyze alternate activities that aid in bacterial adaptation. We previously discovered alternative donors for Escherichia coli DXPS, with donor processing and acceptor specificity influenced by the donor. Here, we demonstrate species differences in DXPS substrate usage and processing which could signify that DXPS homologues support bacterial adaptation in distinct ways. We showed that hydroxypyruvate (HPA) is a donor for Pseudomonas aeruginosa DXPS ( Pa DXPS), similar to Ec DXPS, yet HPA processing and donor–acceptor specificity differs on these homologues; HPA reacts with ThDP on Pa DXPS to form a longer-lived intermediate in the absence of added trigger, relative to Ec DXPS. Notably, Pa DXPS preferentially catalyzes xylulose 5-phosphate formation from HPA and d -GAP, in contrast to Ec DXPS, suggesting a potential connection between DXPS activity and pentose phosphate pathway metabolism in P. aeruginosa . Finally, both Ec DXPS and Pa DXPS were found to generate a 5-carbon β-ketoacid via a one-substrate reaction of HPA, distinguishing DXPS from transketolase (TK) which produces erythrulose under similar conditions. This study illustrates species differences in DXPS donor usage and processing, and donor–acceptor specificity that supports distinct multifunctionality across bacterial pathogens and the plausibility of species-specific targeting.