Publication: Plasmodium falciparum: Asparagine mutant at residue 108 of dihydrofolate reductase is an optimal antifolate-resistant single mutant
1
Issued Date
1997-01-01
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ISSN
00144894
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2-s2.0-0031282471
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Mahidol University
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SCOPUS
Bibliographic Citation
Experimental Parasitology. Vol.87, No.3 (1997), 245-252
Suggested Citation
Worachart Sirawaraporn, Suganya Yongkiettrakul, Rachada Sirawaraporn, Yongyuth Yuthavong, Daniel V. Santi Plasmodium falciparum: Asparagine mutant at residue 108 of dihydrofolate reductase is an optimal antifolate-resistant single mutant. Experimental Parasitology. Vol.87, No.3 (1997), 245-252. doi:10.1006/expr.1997.4221 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/17981
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Title
Plasmodium falciparum: Asparagine mutant at residue 108 of dihydrofolate reductase is an optimal antifolate-resistant single mutant
Abstract
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 Lys108mutant, which was not expressed, all other substitutions yielded DHFR mutants which were expressed in Escherichia coli as inactive inclusion bodies. Nine of the mutants - Asn108, Thr108, Gly108, Ala108, Gln108, Cys108, Val108, Leu108, and Met108- yielded active DHFR upon refolding of the protein from the inclusion bodies. The remaining mutants - Ile108, Arg108Pro108, Asp108, His108, Tyr108, Phe108, and Glu108- did not exhibit detectable DHFR activity on refolding. The Asn108mutant 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 Gln108, Gly108, Cys108, and Ala108mutants. The mutant enzymes had a k(cat)/K(m) values ranging from 7 to 22% of the wild-type enzyme. While DHFRs from Gly108, Cys108, and Ala108mutants were as susceptible to pyrimethamine and cycloguanil as the wild type, the Gln108mutation conferred high resistance to both inhibitors. Our data suggest that residue 108 is important for antifolate binding, and that the Ser108to Asn108mutation 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.