Molecular cloning and characterization of Plasmodium falciparum transketolase.
The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305 microM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.
Joshi
Shweta
S
Division of Biochemistry, Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India.
Singh
Alok Ranjan
AR
Kumar
Ashutosh
A
Misra
Prakash Chandra
PC
Siddiqi
Mohammad Imran
MI
Saxena
Jitendra Kumar
JK
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D016054:DNA, Protozoan / Q000302:isolation & purification; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000166:cytology / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011773:Pyruvates / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D014174:Transketolase / Q000302:isolation & purification* / Q000378:metabolism*
D016054:DNA, Protozoan; D005636:Fructosephosphates; D011773:Pyruvates; D011994:Recombinant Proteins; C027618:fructose-6-phosphate; C012375:hydroxypyruvic acid; D014174:Transketolase