Schöneck R, Billaut-Mulot O, Numrich P, Ouaissi M A, Krauth-Siegel R L
Institut für Biochemie II, Universität Heidelberg, Germany.
Eur J Biochem. 1997 Feb 1;243(3):739-47. doi: 10.1111/j.1432-1033.1997.00739.x.
This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively. An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose. The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.
这项研究展示了来自克氏锥虫（恰加斯病即美洲锥虫病的病原体）的二氢硫辛酰胺脱氢酶（LipDH）的一个cDNA以及两个等位基因的完整序列。全长cDNA有一个1431 bp的开放阅读框，编码一个含477个氨基酸残基的蛋白质。LipDH是一种以FAD为辅基的同二聚体蛋白。结合FAD的成熟蛋白亚基的计算分子量为50,066。克氏锥虫LipDH推导的氨基酸序列与布氏锥虫和人的相比，同一性分别为81%和50%。一个N端九肽（不存在于成熟酶中）代表一种线粒体靶向序列，迄今仅在锥虫中发现。克氏锥虫LipDH的基因lpd1在缺乏LipDH的大肠杆菌JRG1342细胞中无靶向序列表达。为此，在代表成熟蛋白N端的Asn10密码子的直接上游引入了一个ATG密码子。该系统使每1细菌细胞培养物能合成1000 U克氏锥虫LipDH。重组蛋白通过硫酸铵沉淀和5'-AMP-琼脂糖亲和层析纯化至均一。NAD+、NADH、硫辛酰胺和二氢硫辛酰胺的K(m)值与从寄生虫分离的酶相同。Northern和Western印迹分析表明，LipDH存在于克氏锥虫的所有主要发育阶段。这一发现与柠檬酸循环在寄生虫整个生命周期中都活跃一致。对哺乳动物LipDH的体外研究揭示了这种黄素酶催化包括抗锥虫药物硝呋替莫在内的硝基呋喃衍生物的氧化还原循环和超氧阴离子产生的能力。因此，克氏锥虫LipDH被视为基于结构开发新型抗寄生虫药物的一个有前景的靶点。寄生虫酶的细菌表达系统现在将有助于研究克氏锥虫LipDH在药物激活中的作用以及该蛋白的结晶。
