The druggable antimalarial target PfDXR: overproduction strategies and kinetic characterization.
Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) is a key enzyme in the synthesis of isoprenoids in the malaria parasite, using a pathway that is absent in the human host. This enzyme is receiving attention as it has been validated as a promising drug target. However, an impediment to the characterisation of this enzyme has been the inability to obtain sufficient quantities of the enzyme in a soluble and functional form. The expression of PfDXR from the codon harmonised coding region, under conditions of strongly controlled transcription and induction, resulted in a yield of 2-4 mg/L of enzyme, which is 8 to 10-fold higher than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR using an NADPH-dependent assay. Residues K295 and K297, unique to species of Plasmodium and located in the catalytic hatch region; and residues V114 and N115, essential for NADPH binding, were mutated to resemble those found in E. coli DXR. Interestingly, these mutations decreased the substrate affinity of PfDXR to values resembling that of E. coli DXR. PfDXR-K295N, K297S and PfDXR-V114A, N115G demonstrated a decreased ability to turnover substrate by 4-fold and 2-fold respectively in comparison to PfDXR. This study indicates a difference in the role of the catalytic hatch in capturing substrate by species of Plasmodium. The results of this study could contribute to the development of inhibitors of PfDXR.
Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa.
Jessica L
JL
Goble
Hailey
H
Johnson
Jaco
J
de Ridder
Linda L
LL
Stephens
Abraham
A
Louw
Gregory L
GL
Blatch
Aileen
A
Boshoff
D019747:Aldose-Ketose Isomerases / Q000378:metabolism*; D000962:Antimalarials; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism*; D010428:Pentosephosphates / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000737:chemistry / Q000201:enzymology*; D017433:Protein Structure, Secondary
D000962:Antimalarials; D018832:Molecular Chaperones; D010428:Pentosephosphates; C031625:xylulose-5-phosphate; D019747:Aldose-Ketose Isomerases