Peña-Díaz J, Montalvetti A, Camacho A, Gallego C, Ruiz-Perez L M, Gonzalez-Pacanowska D
Instituto de Parasitología y Biomedicina 'López-Neyra', Consejo Superior de Investigaciones Científicas, C/ Ventanilla 11, 18001 Granada, Spain.
Biochem J. 1997 Jun 1;324 ( Pt 2)(Pt 2):619-26. doi: 10.1042/bj3240619.
We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.
我们报告了从恰加斯病的病原体克氏锥虫中分离并鉴定出一个基因组克隆，该克隆包含3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的开放阅读框序列。原生动物基因编码的多肽比其他真核生物中描述的基因所编码的多肽小，推导的氨基酸序列可以与动植物还原酶的C端一半进行比对，与其他真核生物对应物表现出明显的相似性。通过cDNA分析对5'侧翼区域进行进一步检查并确定剪接受体位点，清楚地表明相应的mRNA显然缺乏编码膜N端结构域的序列。还原酶基因是单拷贝的，通过轮廓夹钳均匀电场电泳确定其位于1.36 Mb的染色体上。对克氏锥虫细胞提取物进行差速离心后，研究了酶活性的整体细胞分布。还原酶活性主要与细胞可溶部分相关，因为95%的总细胞活性在上清液中被回收，并且对蛋白水解失活特别敏感。此外，通过使用表达载体pET-11c，该酶可以以高活性形式高效过表达。因此，克氏锥虫HMG-CoA还原酶的独特之处在于它完全缺乏迄今为止所有已鉴定的真核HMG-CoA还原酶中存在的跨膜序列。
