Deletion of the parasite-specific insertions and mutation of the catalytic triad in glutathione reductase from chloroquine-sensitive Plasmodium falciparum 3D7.
The flavoenzyme glutathione reductase (GR; NADPH+glutathione disulphide+H(+)-->NADP(+)+2 glutathione-SH) of Plasmodium falciparum is a promising drug target against tropical malaria. As P. falciparum genes are assumed to be highly polymorphic we have cloned and expressed the GR cDNA of the chloroquine-sensitive strain 3D7. In comparison to the known GR of the chloroquine-resistant K1 strain there are three base exchanges all of them leading to amino acid substitutions (residues 281, 285 and 335). The catalytic efficiency k(cat)/K(m) of the 3D7 enzyme is 5-fold lower than for the K1 enzyme. In contrast, vis-à-vis the drugs carmustine, methylene blue and fluorophenyliso-alloxazine the two enzyme species exhibited identical inhibition kinetics. Two structural motifs which are specific for P. falciparum GR were studied by mutational deletion analysis of 3D7 GR. Loop 126-138 appears to be important for folding and stability of the enzyme, whereas the subdomain 318-350 was found to be involved in FAD-binding. The subdomain has no major influence on the known functions of the catalytic triad Cys-40, Cys-45 and His-485'. Flavin absorption spectroscopy of inactive point mutants showed that Cys-45 forms a thiolate charge transfer complex and Cys-40 is the interchange thiol, which reduces glutathione disulphide. The mutant His-485-->Gln had a normal K(m) for glutathione disulphide reduction but only 0.8% residual catalytic activity when compared with wild-type GR, which confirms its function as an acid/base catalyst. The parasite-specific domains in combination with the reactive catalytic residues appear to be a suitable target matrix for inhibiting GR in vivo.
Gilberger
T W
TW
Bernhard Nocht Institute for Tropical Medicine, Biochemical Parasitology, Bernhard-Nocht-Strasse 74, D-20359, Hamburg, Germany.
Schirmer
R H
RH
Walter
R D
RD
Müller
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D002384:Catalysis; D002738:Chloroquine / Q000494:pharmacology*; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D017354:Point Mutation; D013057:Spectrum Analysis / Q000379:methods
D000962:Antimalarials; D005415:Flavins; D002738:Chloroquine; D005980:Glutathione Reductase