Allen T E, Ullman B
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
Mol Biochem Parasitol. 1994 Jun;65(2):233-45. doi: 10.1016/0166-6851(94)90075-2.
The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme in Trypanosoma cruzi is a rational target for the treatment of Chagas disease. To evaluate the T. cruzi HGPRT in detail, the HGPRT gene (hgprt) was cloned from a genomic library of T. cruzi DNA and sequenced. Translation of the nucleotide sequence of the hgprt revealed an open reading frame of 663 bp that encoded a 25.5-kDa polypeptide of 221 amino acids. The T. cruzi HGPRT exhibited only 24%, 25%, and 21% amino acid sequence identity to its human, Plasmodium falciparum, and Schistosoma mansoni counterparts, respectively, but was 50% identical to the T. brucei HGPRT protein. Northern analysis of T. cruzi RNA revealed a 1.8-kb hgprt transcript, while Southern blots of genomic DNA suggested that hgprt was a single copy gene within the T. cruzi genome. The T. cruzi hgprt was inserted into the pBAce expression plasmid and transformed into Escherichia coli that are deficient in hypoxanthine and guanine phosphoribosylating activities. High levels of soluble, enzymatically active T. cruzi HGPRT were obtained, and this expression complemented the bacterial phosphoribosyltransferase deficiencies. The recombinant HGPRT was purified to apparent homogeneity by GTP-agarose affinity chromatography and recognized hypoxanthine, guanine, and allopurinol, but not adenine or xanthine, as substrates. The availability of the hgprt clone and large amounts of pure HGPRT protein provide a foundation for a structure-based drug design strategy for the treatment of Chagas disease.
克氏锥虫中的次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HGPRT）是治疗恰加斯病的合理靶点。为了详细评估克氏锥虫HGPRT，从克氏锥虫DNA基因组文库中克隆了HGPRT基因（hgprt）并进行测序。hgprt核苷酸序列的翻译揭示了一个663 bp的开放阅读框，其编码一个由221个氨基酸组成的25.5 kDa多肽。克氏锥虫HGPRT与其人类、恶性疟原虫和曼氏血吸虫对应物的氨基酸序列同一性分别仅为24%、25%和21%，但与布氏锥虫HGPRT蛋白有50%的同一性。对克氏锥虫RNA的Northern分析揭示了一个1.8 kb的hgprt转录本，而基因组DNA的Southern印迹表明hgprt是克氏锥虫基因组中的单拷贝基因。将克氏锥虫hgprt插入pBAce表达质粒并转化到缺乏次黄嘌呤和鸟嘌呤磷酸核糖化活性的大肠杆菌中。获得了高水平的可溶性、具有酶活性的克氏锥虫HGPRT，并且这种表达弥补了细菌磷酸核糖转移酶的缺陷。通过GTP - 琼脂糖亲和层析将重组HGPRT纯化至表观均一，并且该重组酶识别次黄嘌呤、鸟嘌呤和别嘌呤醇作为底物，但不识别腺嘌呤或黄嘌呤。hgprt克隆的可得性和大量纯HGPRT蛋白为基于结构的恰加斯病治疗药物设计策略提供了基础。
