Kuramochi T, Hirawake H, Kojima S, Takamiya S, Furushima R, Aoki T, Komuniecki R, Kita K
Department of Parasitology, University of Tokyo, Japan.
Mol Biochem Parasitol. 1994 Dec;68(2):177-87. doi: 10.1016/0166-6851(94)90163-5.
Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.
寄生线虫猪蛔虫成虫线粒体中的复合物II具有较高的延胡索酸还原酶活性，在这些细胞器中观察到的厌氧电子传递中起关键作用。在本研究中，已从猪蛔虫和需氧的自由生活线虫秀丽隐杆线虫中分离、克隆并测序了复合物II黄素蛋白（Fp）亚基的cDNA。通过cDNA末端快速扩增（RACE）确定了mRNA 3'末端的额外序列。猪蛔虫cDNA的核苷酸序列分析揭示了许多线虫mRNA特有的22个核苷酸的反式剪接前导序列、1935个核苷酸的开放阅读框和616个核苷酸的3'非翻译区，包括来自聚腺苷酸化信号（AATAAA）的聚（A）尾。开放阅读框编码一个645个氨基酸的序列，包括一个30个氨基酸的线粒体前序列。尽管蛔虫酶在生理上作为延胡索酸还原酶发挥作用，而秀丽隐杆线虫酶作为琥珀酸脱氢酶发挥作用，但两种生物的Fp亚基的氨基酸序列非常相似。蛔虫序列与大肠杆菌延胡索酸还原酶的相似性要低得多。其他Fp亚基对巯基试剂的敏感性似乎存在于假定活性位点中保守精氨酸之前的一个半胱氨酸处。在两个线虫序列中，尽管两种酶的琥珀酸脱氢酶活性仍然对巯基抑制敏感，但这个半胱氨酸被丝氨酸取代。丝氨酸上游六个残基处的一个半胱氨酸可能与线虫酶的巯基敏感性有关。令人惊讶的是，与琥珀酸脱氢酶活性相反，蛔虫酶的延胡索酸还原酶活性对巯基抑制不敏感，这表明这两个反应的机制涉及不同的催化过程。
