The Y392F further stabilizes the pre‐decarboxylation intermediate on 1‐deoxy‐D‐xylulose 5‐phosphate synthase

Abstract

The thiamin diphosphate (ThDP)‐dependent enzyme 1‐deoxy‐D‐xylulose 5‐phosphate (DXP) synthase catalyzes the condensation of D‐glyceraldehyde‐3‐phosphate (D‐GAP) and pyruvate to form DXP, a precursor in the biosynthesis of vitamins B1, B6 and isoprenoids. Our groups recently reported (J.Am.Chem.Soc. 2012, 134, 18374–18379) stabilization of the ThDP‐bound predecarboxylation intermediate C2α‐lactylThDP on DXP synthase and an at least 600‐fold rate acceleration of the decarboxylation by D‐GAP. Model building and analogy with similar enzymes suggested that the residue Y392 could be responsible for this finding, perhaps by hydrogen bonding to the carboxylate anion of C2α‐lactylThDP. To explore this hypothesis, we have carried out steady state and pre‐steady state experiments with the DXP synthase Y392F variant. The results suggest that C2α‐lactylThDP is even more stable on the variant than on DXP synthase. An NMR experiment of the reaction mixture containing Y392F and pyruvate after 50s acid quench also supports the idea that Y392F stabilizes C2α‐lactylThDP. In the presence of D‐GAP the rate of decarboxylation was similar to that on DXP synthase, suggesting that Y392 might be holding the carboxylate group of C2α‐lactylThDP and addition of acceptor substrate D‐GAP immediately rescues the slow rate of decarboxylation. Supported by NIHGM050380 (RU) and NIH‐GM084998 (JHU).