Plasmodium falciparum: Asparagine mutant at residue 108 of dihydrofolate reductase is an optimal antifolate-resistant single mutant

Plasmodium falciparum: Asparagine mutant at residue 108 of dihydrofolate reductase is an optimal antifolate-resistant single mutant. Experimental Parasitology 87, 245-252. The codon for serine residue 108 of the Plasmodium falciparum dihydrofolate reductase gene was replaced with those for the other 19 amino acids. Except for the Lys(108) mutant, which was not expressed, all other substitutions yielded DHFR mutants which were expressed in Escherichia coli as inactive inclusion bodies. Nine of the mutants-Asn(108), Thr(108), Gly(108), Ala(108), Gln(108), Cys(108), Val(108), Leu(108), and Met(108) yielded active DHFR upon refolding of the protein from the inclusion bodies. The remaining mutants-Ile(108), Arg(108) Pro(108), Asp(108), His(108), Tyr(108), Phe(108), Trp(108), and Glu(108)-did nor exhibit detectable DHFR activity on refolding. The Asn(108) mutant had almost unperturbed kinetic parameters but conferred resistance to pyrimethamine and cycloguanil; other active mutants showed poorer DHFR activity. We purified and characterized four mutants which produced highest DHFR activity, i.e., the Gln(108), Glp(108), Cys(108), and Ala(108) mutants. These mutant enzymes had k(cat)/K-m values ranging from 7 to 22% of the wild-type enzyme. While DHFRs from Gly(108), Cys(108), and Ala mutants were as susceptible to pyrimethamine and cycloguanil as the wild type, the Gln(108) mutation conferred high resistance to both inhibitors. Our data suggest that residue 108 is important for antifolate binding, and that the Ser(108) to Asn(108) mutation was selected in nature because of (i) the need for only a single base change, (ii) its good activity, and (iii) its resistance to antifolates. (C) 1997 Academic Press.