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

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 inhibitio...

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Bibliographic Details
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
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Summary: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.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2015.12.003