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The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.

Emerging resistance of human pathogens to anti-infective agents make it necessary to develop new agents to treat infection. The methylerythritol phosphate pathway has been identified as an anti-infective target, as this essential isoprenoid biosynthetic pathway is widespread in human pathogens but absent in humans. The first enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the formation of DXP via condensation of D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate in a thiamine diphosphate-dependent manner. Structural analysis has revealed a unique domain arrangement suggesting opportunities for the selective targeting of DXP synthase; however, reports on the kinetic mechanism are conflicting. Here, we present the results of tryptophan fluorescence binding and kinetic analyses of DXP synthase and propose a new model for substrate binding and mechanism. Our results are consistent with a random sequential kinetic mechanism, which is unprecedented in this enzyme class.

A study of DXP synthase has revealed flexibility in the acceptor substrate binding pocket for nonpolar substrates and has uncovered new details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate.

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.