The rapid development of antimicrobial resistance underscores the need for novel antibiotics. The unexploited drug target 1-deoxy-D-xyluose synthase (DXPS) catalyzes the thiamine diphosphate (ThDP)-dependent formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GAP). DXP is an intermediate in the biosynthesis of ThDP, pyridoxal phosphate, and isoprenoids in bacteria but not in humans. Thus, selective inhibition of this branch point enzyme is a potential antibacterial strategy. Previous studies show that DXPS possesses relaxed specificity for its acceptor substrate and proceeds through a unique random sequential, preferred order mechanism. GAP serves two roles, 1) as the trigger for decarboxylation of the first enzyme-bound intermediate lactylThDP (LThDP), stabilized by DXPS in the absence of a trigger, and 2) as acceptor in the carboligation step. This unprecedented mechanism in ThDP enzymology implies distinct DXPS conformations exist along the reaction coordinate which could offer opportunities for selective inhibitor design and hints at the possibility that DXPS could possess other cellular functions. Studies aimed at understanding factors underlying DXPS stabilization of LThDP and GAP-induced decarboxylation have revealed oxygenase activity of DXPS, an unexpected finding given the observation of LThDP accumulation on DXPS and lack of evidence for post-decarboxylation intermediates in the absence of GAP. Here we present evidence for oxygenase activity of DXPS under conditions where LThDP was previously observed and new insights into triggers of LThDP decarboxylation. Our results suggest that LThDP decarboxylation takes place in an O<sub>2</sub>-dependent manner in the absence of GAP and peracetate is detected as an intermediate in acetate formation. The implications of these findings for DXPS mechanism will be discussed. The identification of distinct triggers of LThDP on DXPS decarboxylation combined with a remarkable flexibility for the acceptor substrate lends further intrigue and support to the notion that this novel mechanism may impart useful, yet uncharacterized cellular functions to DXPS.

Investigating the mechanism of LThDP decarboxylation by DXP synthase

254th National Meeting (2017)

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