Pandini Vittorio, Caprini Gianluca, Thomsen Nadine, Aliverti Alessandro, Seeber Frank, Zanetti Giuliana
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
J Biol Chem. 2002 Dec 13;277(50):48463-71. doi: 10.1074/jbc.M209388200. Epub 2002 Oct 4.
Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.
刚地弓形虫拥有一种定位于顶质体的植物型铁氧化还原蛋白-NADP(+)还原酶。我们从同一寄生虫中克隆了一种[2Fe-2S]铁氧化还原蛋白，以研究这两种氧化还原蛋白之间的相互作用。已对两种纯化的重组蛋白进行了详细表征，特别是关于它们之间的相互作用。该双蛋白复合物能够高效催化电子从NADPH转移至细胞色素c。结果表明，还原酶的黄素辅因子（FAD/FADH(-)）的氧化还原电位比NADP(+)/NADPH电对的更正，因此有利于电子从NADPH转移以产生还原型铁氧化还原蛋白。通过几种方法（酶活性、交联、蛋白质荧光猝灭、亲和色谱）在体外以及通过酵母双杂交系统在体内研究了还原酶与来自各种来源的铁氧化还原蛋白之间的复合物形成。我们的数据表明，这两种蛋白形成了具有高亲和力的活性复合物，强烈表明刚地弓形虫的这两种蛋白形成了一个生理氧化还原对，该对将电子从NADPH转移至铁氧化还原蛋白，而铁氧化还原蛋白又被顶质体的某些还原性生物合成途径所利用。这些数据为探索这一氧化还原对作为顶复门寄生虫的药物靶点提供了基础。
