Duran E, Walker D J, Johnson K R, Komuniecki P R, Komuniecki R W
Department of Biology, University of Toledo, OH 43606-3390, USA.
Mol Biochem Parasitol. 1998 Mar 15;91(2):307-18. doi: 10.1016/s0166-6851(97)00212-0.
The 2-methyl branched-chain enoyl CoA reductase (ECR) plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode, Ascaris suum. Two-dimensional gel electrophoresis of the purified ECR yielded multiple spots, with two distinct but overlapping N-terminal sequences. These multiple isoforms were not the result of population effects, as the pattern observed on 2-D gels of the purified ECR was identical to those on immunoblots of muscle homogenates isolated from individual worms. A full-length cDNA coding for the major ECR isoform (ECRI) has been cloned and sequenced and compared with that of the minor isoform (ECRII) which has been described previously (Duran et al. J Biol Chem 1993;268:22391-22396). ECRI contained the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a 5' untranslated region (UTR) of 13 nucleotides, an open reading frame (ORF) of 1257 nucleotides, a 3'-UTR of 110 nucleotides that included the polyadenylation signal AATAAA downstream of the termination codon and a short poly(A) tail. The ORF predicted a 16 amino acid leader sequence not found in the native protein and a mature protein of 403 amino acids with a molecular weight of 43 698 and a predicted pI of 6.2. ECRI and ECRII were 73% identical at the predicted amino acid level and their mRNAs exhibited significant structural similarity even though they were products of separate genes. Comparison of ECRI and ECRII with the sequences of acyl CoA dehydrogenases from a variety of different sources revealed a high degree of interspecies sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene. This result is surprising since the ascarid enzymes function as reductases, not as dehydrogenases. Both ECRs were tissue-specific and developmentally regulated and were found in transitional third-stage larvae (L3) and adult muscle, but not in early, aerobic larval stages or adult testis, ovary, or intestine. The ratio of ECRII to ECRI was greater in L3 than in adult muscle. Interestingly, both ECRs also appeared to be expressed in pharyngeal muscle, suggesting that branched-chain fatty acid synthesis may not be confined exclusively to body wall muscle.
2-甲基支链烯酰辅酶A还原酶（ECR）在寄生线虫猪蛔虫厌氧线粒体中进行的β-氧化逆转过程中起着关键作用。纯化后的ECR经二维凝胶电泳产生多个斑点，具有两个不同但重叠的N端序列。这些多种同工型并非群体效应的结果，因为在纯化ECR的二维凝胶上观察到的模式与从单个蠕虫分离的肌肉匀浆免疫印迹上的模式相同。编码主要ECR同工型（ECRI）的全长cDNA已被克隆、测序，并与先前描述的次要同工型（ECRII）进行了比较（杜兰等人，《生物化学杂志》，1993年；268:22391 - 22396）。ECRI包含许多线虫mRNA特有的22个核苷酸的反式剪接前导序列、13个核苷酸的5'非翻译区（UTR）、1257个核苷酸的开放阅读框（ORF）、110个核苷酸的3'-UTR，该区域在终止密码子下游包含多聚腺苷酸化信号AATAAA和一个短的聚（A）尾巴。该ORF预测有一个在天然蛋白质中未发现的16个氨基酸的前导序列和一个由403个氨基酸组成的成熟蛋白质，分子量为43698，预测的pI为6.2。ECRI和ECRII在预测的氨基酸水平上有73%的同一性，并且它们的mRNA即使是不同基因的产物也表现出显著的结构相似性。将ECRI和ECRII与来自各种不同来源的酰基辅酶A脱氢酶序列进行比较，发现种间序列同一性程度很高，这表明这些酶可能是从一个共同的祖先基因进化而来的。这个结果令人惊讶，因为蛔虫的这些酶作为还原酶起作用，而不是脱氢酶。两种ECR都是组织特异性的且受发育调控，在过渡性第三期幼虫（L3）和成虫肌肉中发现，但在早期需氧幼虫阶段或成虫的睾丸、卵巢或肠道中未发现。L3中ECRII与ECRI的比例大于成虫肌肉中的比例。有趣的是，两种ECR似乎也在咽肌中表达，这表明支链脂肪酸的合成可能并不局限于体壁肌肉。
