Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi.
Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi.
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人类病原体克氏锥虫二氢硫辛酰胺脱氢酶的克隆、测序和功能表达。
DOI：
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
R. Luke Krauth
中科院分区：
文献类型：
--
作者：
R. Schoneck;O. Billaut‐Mulot;Petra Numrich;M. A. Ouaissi;R. Luke Krauth
本文报道了查加斯病(美国锥虫病)病原体--克氏锥虫二氢硫胺脱氢酶基因的全序列和两个等位基因。该基因全长为1431bp，编码477个氨基酸残基。LipDh是一种以FAD为辅基的同源二聚体蛋白。计算出与Fad结合的成熟蛋白质亚基的分子质量为50,066。克氏锥虫和布氏锥虫、人脂脱氢酶的氨基酸序列同源性分别为81%和50%。在成熟的酶中不存在的N-末端九肽，代表了迄今为止只在锥虫中发现的线粒体靶向序列。克氏毛滴虫LipDh基因lpd1在缺失LipDh的大肠杆菌JRG1342细胞中表达，无靶向序列。为此，在Asn10密码子的上游直接引入了ATG密码子，Asn10代表成熟蛋白质的N端。该体系可用于合成1000U克鲁兹杆菌LipDH/1细菌细胞培养物。重组蛋白经(NH_4)_2SO_4沉淀和5‘AMP-Sepharose亲和层析纯化。NAD+、NADH、硫胺和二氢硫胺的K(M)值与从寄生虫中分离的酶的K(M)值一致。Northern和Western印迹分析表明，脂肪脱氢酶存在于克氏锥虫的所有主要发育阶段。这一发现与寄生虫整个生命周期中活跃的柠檬酸循环是一致的。对哺乳动物脂肪脱氢酶的体外研究表明，该黄素酶能够催化硝基呋喃衍生物的氧化还原循环和超氧阴离子的产生，其中包括抗锥虫药物硝呋莫司。因此，克氏毛滴虫LipDH被认为是基于结构的新型抗寄生虫药物开发的一个很有前途的靶点。这种寄生虫酶的细菌表达系统现在将允许研究克氏锥虫脂肪水解酶在药物激活和蛋白质结晶中的作用。
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.
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影响因子：
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作者：
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发表时间：
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期刊：
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DOI：
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发表时间：
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期刊：
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DOI：
10.1006/prep.1995.1011
发表时间：
1995
期刊：
Protein expression and purification.
影响因子：
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作者：
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锥虫 Rieske 铁硫蛋白具有可能指导线粒体输入的切割前序列。
DOI：
10.1016/0167-4889(95)00154-6
发表时间：
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期刊：
Biochimica et biophysica acta
影响因子：
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