Diaz-González Rosario, Pérez-Pertejo Yolanda, Pommier Yves, Balaña-Fouce Rafael, Reguera Rosa M
Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain.
Biochem Pharmacol. 2008 Sep 1;76(5):608-19. doi: 10.1016/j.bcp.2008.06.019. Epub 2008 Jul 4.
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.
杜氏利什曼原虫是内脏利什曼病的病原体，它含有一种独特的双亚基DNA拓扑异构酶IB（LdTopIB）。具有催化活性的酶是一种异源二聚体，由一个大亚基（LdTopIL）和一个小亚基（LdTopIS）组成。大亚基包含一个非保守的N末端和系统发育保守的核心结构域，而小亚基在活性位点带有具有特征性酪氨酸残基的C末端结构域。采用定点诱变将LdTopIL亚基的碱性氨基酸（Arg-314、Lys-352、Arg-410和His-453）替换为中性氨基酸丙氨酸。这些突变体在无拓扑异构酶的酵母菌株中的表达产生了无活性的蛋白质。同样，当参与DNA切割的小亚基中的Tyr-222被Phe取代时，在过表达提取物的酵母中未检测到拓扑异构酶活性。此外，还分析了与喜树碱抑制有关的两个替换。位于大亚基核心结构域的Asp-353和在小亚基中位于Tyr-222上游的Asn-221分别被Ala和Ser取代。这些突变体对抑制剂不敏感；尽管它们表现出显著的松弛活性。
