Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue
Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue
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DOI：
10.1021/bi952998u
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发表时间：
1996-05-14
期刊：
影响因子：
2.9
通讯作者：
Schramm, VL
中科院分区：
文献类型：
--
作者：
Gopaul, DN;Meyer, SL;Schramm, VL
原生动物依赖嘌呤补救来合成核酸。束状短翅虫中一种丰富的补救酶是肌苷-尿苷核苷水解酶（IU-核苷水解酶）。用与胰蛋白酶片段的氨基酸序列和C.束状。在大肠杆菌中表达全长cDNA，并将蛋白纯化至>99%同源性。开放阅读框编码315个氨基酸的蛋白质。从C. fasciculata缺失N-末端Met，质谱分析显示其主要质量峰为34 194 amu。Met处理的酶的DNA序列的预测质量为34 196。将pET 3d-IUNH构建体在E.大肠杆菌在N-末端引入MetAla而不是MetPro。从该构建体纯化的酶也具有加工的N-末端，并且分别给出34 168和34 170 amu的预测和观察质量。IU-核苷水解酶的氨基酸序列在已知蛋白质中没有近亲。硕大利什曼原虫的一个未知功能的cDNA克隆在N-末端推导的氨基酸序列中显示出接近的同一性。在E上打开1分钟和47分钟附近的阅读框。大肠杆菌染色体和两个酵母基因组编码大小相似且具有实质性氨基酸同一性的蛋白质。His 241 Ala的突变导致肌苷的k（cat）损失2100倍，但对硝基苯基β-D-呋喃核糖苷的k（cat）增加2.8倍，建立了催化位点的位置，并暗示His 241是离去基团活化的质子供体。IU-核苷水解酶。fasciculata和E.大肠杆菌中的蛋白质分别结晶和纯化至2.5和2.1埃分辨率。两者都属于P2（1）2（1）2正交空间群，晶胞参数a = 63.5埃，B = 131.9埃，c = 90.1埃，α = β = γ = 90度。四聚体酶的两个亚基存在于不对称单元中。下面的论文报道了这种酶的X射线晶体结构。
Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to >99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 Angstrom resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 Angstrom, b = 131.9 Angstrom, c = 90.1 Angstrom, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.