Abstract
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.
Highlights
Members of the isoprenoid natural product class are constructed from the precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which are essential in all living organisms
The natural substrate for this reaction, 1-deoxy-D-xylulose 5-phosphate (DXP),4 is generated from pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) in a thiamine diphosphate (ThDP)-dependent reaction catalyzed by DXP synthase [5]
The reaction catalyzed by DXP synthase combines elements of ThDP-dependent decarboxylase and carboligase chemistry [5], and active site residues necessary for coordinating the cofactor are conserved relative to other ThDP-dependent enzymes [10]
Summary
Work on the kinetic mechanism of Rhodobacter capsulatus DXP synthase suggests this enzyme is mechanistically distinct from other ThDP-dependent enzymes [12] despite the similar carboligation transformation it catalyzes. All other ThDP-dependent enzymes are believed to operate via a classical ping-pong mechanism [11], whereas CO2 trapping studies performed by Eubanks and Poulter [12] have provided compelling evidence for the requirement of a ternary complex in DXP synthase catalysis. Using immobilized enzyme and substrate, the authors determined substrate binding constants and suggested that the observed 1.7-fold enhancement in binding of D-GAP to DXP synthase in the presence of soluble pyruvate provides further support for an ordered mechanism. A conflicting report [15] proposes, on the basis of steady-state kinetics, that Escherichia coli and Haemophilus influenzae DXP synthase operate via a classical ping-pong mechanism in a manner similar to other ThDP-dependent enzymes (Fig. 2B). We have performed detailed kinetic analyses using an unreactive pyruvate analog, methylacetylphosphonate (MAP), as a reversible inhibitor to elucidate substrate binding in the reaction catalyzed by DXP synthase. Our results indicate that D-GAP and pyruvate bind independently and reversibly to DXP synthase, suggesting the enzyme proceeds via a novel rapid equilibrium random sequential mechanism
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