Yuan L, Craig S P, McKerrow J H, Wang C C
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
J Biol Chem. 1990 Aug 15;265(23):13528-32.
Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.
由于缺乏嘌呤核苷酸的从头生物合成，次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HGPRTase）是人体寄生虫曼氏血吸虫中供应鸟嘌呤核苷酸的必需酶，并且已被提议作为抗寄生虫化疗的潜在靶点。虽然该酶可以从成年血吸虫中纯化出来，但产量太低，无法进行广泛的结构和动力学研究。因此，我们克隆并测序了编码血吸虫酶的cDNA和基因，但无法从DNA序列中准确鉴定该酶的氨基末端序列。在尝试准确表达活性酶之前，了解确切的氨基末端是必要的。因此，我们从成年虫的粗提物中纯化了HGPRTase。纯化后的酶亚基分子量为26 kDa，氨基末端序列为Met-Ser-Ser-Asn-Met。该序列与从cDNA和基因序列预测的潜在起始位点之一相匹配。接下来，我们使用受细菌碱性磷酸酶启动子调控的载体，并利用大肠杆菌信号肽将表达产物分泌到周质空间，在大肠杆菌中表达曼氏血吸虫HGPRTase正确大小的cDNA。使用该表达系统，一些重组酶被分泌出来，并且发现其氨基末端正确。留在细胞质中的重组酶氨基末端连接有部分信号肽。从转化大肠杆菌周质中分离出的重组血吸虫HGPRTase经过纯化，发现其动力学和物理性质与天然酶相同。
