Mutational study of the "catalytic tetrad" of DNA topoisomerase IB from the hemoflagellate Leishmania donovani: Role of Asp-353 and Asn-221 in camptothecin resistance.
Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique bisubunit DNA-topoisomerase IB (LdTopIB). The catalytically active enzyme is a heterodimer constituted by a large subunit (LdTopIL) containing a non-conserved N-terminal end and the phylogenetically conserved core domain, whereas the small subunit (LdTopIS) harbors the C-terminal domain with the characteristic tyrosine residue in the active site. Site-directed mutagenesis was used to substitute the basic amino acid (Arg-314, Lys-352, Arg-410 and His-453) of the LdTopIL subunit by the neutral amino acid alanine. The expression of these mutants in a topoisomerase-free yeast strain produced inactive proteins. Similarly, when the Tyr-222 from small subunit, involved in DNA cleavage, was substituted by Phe no topoisomerase activity was detected in yeast overexpressing extracts. In addition two substitutions involved in camptothecin inhibition were also analyzed. Asp-353 located in the core domain of the large subunit and Asn-221 which heads Tyr-222 in the small subunit, were replaced by Ala and Ser, respectively. These mutants were insensitive to the inhibitor; despite they displayed significant relaxation activity.
Diaz-González
Rosario
R
Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain.
Pérez-Pertejo
Yolanda
Y
Pommier
Yves
Y
Balaña-Fouce
Rafael
R
Reguera
Rosa M
RM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005407:Flagella / Q000201:enzymology; D007893:Leishmania donovani / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D059004:Topoisomerase I Inhibitors
D017931:DNA Primers; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D059004:Topoisomerase I Inhibitors; D001224:Aspartic Acid; D001216:Asparagine; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin