Monzani Paulo S, Alfonzo Juan D, Simpson Larry, Oliva Glaucius, Thiemann Otavio H
Laboratory of Protein Crystallography and Structural Biology, Physics Institute of São Carlos, University of São Paulo-USP, Av. Trabalhador Sãocarlense 400, São Carlos, Brazil.
Biochim Biophys Acta. 2002 Jul 29;1598(1-2):3-9. doi: 10.1016/s0167-4838(02)00334-5.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae hgprt gene contains a 633-nucleotide open reading frame that encodes a 23.4-kDa protein. A pairwise alignment of the different HGPRT's sequences revealed a 26%-53% sequence identity with the Leishmania sequences and 87% identity to the HGPRT of Leishmania donovani. A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The steady-state kinetic parameters were determined for the recombinant enzyme and the enzyme is active as a homodimer in solution. Single crystals were obtained for the L. tarentolae HGPRT representing the first Leishmania HGPRT crystallized and initial crystallographic data were collected. The crystals obtained belong to the orthorhombic space group (P2(1)2(1)2(1)) with unit cell parameters a=58.104 A, b=85.443 A and c=87.598 A and diffract to a resolution of 2.3 A. The availability of the HGPRT enzyme from Leishmania and its crystallization suitable for X-ray diffraction data collection should provide the basis for a functional and structural analysis of this enzyme, which has been proposed as a potential target for rational drug design, in a Leishmania model system.
次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HGPRT）（EC 2.4.2.8）是一种参与所有细胞中嘌呤核苷酸循环利用的重要酶。动质体目寄生原生动物无法从头合成嘌呤，而是利用补救途径合成核苷酸；因此，该途径是抗寄生虫药物设计的一个有吸引力的靶点。从一株热带利什曼原虫基因组文库中克隆了hgprt基因并测定了其序列。热带利什曼原虫hgprt基因包含一个633个核苷酸的开放阅读框，编码一个23.4 kDa的蛋白质。对不同HGPRT序列的两两比对显示，与利什曼原虫序列的序列同一性为26% - 53%，与杜氏利什曼原虫的HGPRT的同一性为87%。在大肠杆菌中表达了一种重组蛋白，纯化至同质，并发现其保留了酶活性。测定了重组酶的稳态动力学参数，该酶在溶液中作为同二聚体具有活性。获得了代表首个结晶的利什曼原虫HGPRT的热带利什曼原虫HGPRT单晶，并收集了初始晶体学数据。所获得的晶体属于正交晶系空间群（P2(1)2(1)2(1)），晶胞参数a = 58.104 Å，b = 85.443 Å，c = 87.598 Å，衍射分辨率为2.3 Å。来自利什曼原虫的HGPRT酶及其适合X射线衍射数据收集的结晶，应为在利什曼原虫模型系统中对该酶进行功能和结构分析提供基础，该酶已被提议作为合理药物设计的潜在靶点。
