STAFF PRIORITY TIME XRAY STRUCT ANALYSIS OF INOSINEMONOPHOSPHATE DEHYDROGENASE

肌苷单磷酸脱氢酶(IMPDH)催化 依赖NAD的肌苷一磷酸(IMP)氧化生成黄嘌呤 单磷酸盐(XMP)。这是嘌呤的限速步骤。 生物合成。这种酶的抑制剂已经被证明有 抗肿瘤、免疫抑制和抗寄生虫作用。抑制 在体外抑制了生物体的生长，并 这种抑制可以通过添加GMP来克服。 原始数据、衍生数据和复杂数据在SSRL以7：1收集。 良好的硫柳汞、PCMBS和四氯铂酸盐衍生数据集 并将其用于MIR定相。初始低分辨率SIR 而MIR电子密度图显示了一个扁平的四聚体分子， 与预期的水晶填料一致，其中有一个503 不对称单元中的氨基酸单体，四聚体为 由结晶学四重运算符生成。每种单体 形成一个与三糖的a/b桶非常相似的a/b桶 磷酸异构酶(Tim Barrel)。后续阶段组合： MIR和改进的部分模型阶段使我们能够适应大多数 氨基酸主链和侧链。重原子差图 清楚地标明5个半胱氨酸残基与PCMBS或 硫柳汞和3个与氯化铂结合的蛋氨酸残基。 活动站点已由活动站点的位置标识 半胱氨酸与电子密度在差图中的位置 根据收集的关于配体和抑制剂浸泡的数据进行计算 水晶。

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the NAD-dependent oxidation of inosine monophosphate (IMP) to xanthine monophosphate (XMP). This is the rate-limiting step in purine biosynthesis. Inhibitors of this enzyme have been shown to have anti-tumor, immunosuppressive, and antiparasitic effects. Inhibition of T. foetus IMPDH in vitro arrests the growth of the organism and this inhibition can be overcome by supplementing the medium with GMP. Native, derivative, and complex data were collected at 7-1 at SSRL. Good thimerosal, PCMBS and tetra-chloro-platinate derivative data sets were obtained and used for MIR phasing. Initial low-resolution SIR and MIR electron density maps indicated a flat, tetrameric molecule, consistent with the expected crystal packing where there is one 503 amino-acid monomer in the asymmetric unit, and the tetramer is generated by the crystallographic four-fold operator. Each monomer forms an a/b barrel resembling very closely the a/b barrel of triose phosphate isomerase (TIM barrel). Subsequent phase combination with the MIR and refined partial model phases has allowed us to fit most of the amino acid backbone and side chains. Heavy-atom difference maps indicate clearly 5 cysteine residues that reacted with either PCMBS or thimerosal, and 3 methionine residues that bound platinum chloride. The active site has been identified by the position of an active-site cysteine and the positions of electron density in difference maps calculated from data collected on ligand and inhibitor-soaked crystals.