Mechanistic binding insights for 1-deoxy-d-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax

• Published in: Protein expression and purification 2016-04, Vol.120, p.16-27
• Main Authors: Battistini, Matthew R., Shoji, Christopher, Handa, Sumit, Breydo, Leonid, Merkler, David J.
• Format: Article
• Language: English
• Subjects: 1-Deoxy-d-xylulose-5-phosphate synthase; Amino Acid Sequence; Catalytic Domain; Cloning, Molecular; Glyceraldehyde 3-Phosphate - metabolism; Isoprenoids; Kinetics; Malaria; Methylerythritol phosphate pathway; Molecular Sequence Data; Plasmodium falciparum; Plasmodium falciparum - enzymology; Plasmodium vivax; Plasmodium vivax - enzymology; Protozoan Proteins - metabolism; Pyruvic Acid - metabolism; Recombinant Proteins - metabolism; Sequence Alignment; Transferases - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.12.003

We have successfully truncated and recombinantly-expressed 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and d-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria. •The novel truncation and expression of Plasmodium falciparum DXS and Plasmodium vivax DXR.•Enzymatic characterization by steady-state kinetics, inhibition, and binding constants.•Confirmed previous reports of a random sequential binding mechanism for both P. falciparum and P. vivax DXS.