Cryptosporidium parvum IMP dehydrogenase -: Identification of functional, structural, and dynamic properties that can be exploited for drug design
Cryptosporidium parvum IMP dehydrogenase -: Identification of functional, structural, and dynamic properties that can be exploited for drug design
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DOI：
10.1074/jbc.m407121200
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发表时间：
2004-09-24
影响因子：
4.8
通讯作者：
Striepen, B
中科院分区：
文献类型：
--
作者：
Umejiego, NN;Li, C;Striepen, B
原生动物微小隐孢子虫引起严重的肠炎，在艾滋病患者和幼儿中具有相当高的发病率和死亡率。目前还没有完全有效的治疗方法。微小隐孢子虫依赖肌苷5‘-单磷酸脱氢酶(IMPDH)产生鸟嘌呤核苷酸，对IMPDH抑制高度敏感。此外，微小隐孢子虫通过侧向转移获得了IMPDH基因，这表明寄生虫酶可能具有与人类对应酶非常不同的特性。在这里，我们描述了重组微小弧菌IMPDH在缺乏细菌同源物的大肠杆菌菌株中的表达。寄生虫基因的表达使该突变株在微量培养基上恢复生长，证实该蛋白具有IMPDH活性。重组蛋白纯化至均一，并用于一系列动力学实验中对该酶的作用机制、结构和抑制率的研究。细小葡萄球菌酶的作用机制包括随机添加底物和有序释放产物，并对共价酶中间体进行限速水解。微小葡萄球菌IMPDH对霉酚酸抑制的明显抗性与其细菌来源有很强的一致性。NAD的K-m值和麦考酚酸的K-I值以及噻唑呋喃和ADP之间的协同作用与人的酶有明显的不同。这些数据表明，微小隐孢子虫IMPDH的NAD结合位点的结构和动态特性可以被用来开发寄生虫特异性抑制剂。
The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children. No fully effective treatment is available. C. parvum relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to produce guanine nucleotides and is highly susceptible to IMPDH inhibition. Furthermore, C. parvum obtained its IMPDH gene by lateral transfer from an epsilon-proteobacterium, suggesting that the parasite enzyme might have very different characteristics than the human counterpart. Here we describe the expression of recombinant C. parvum IMPDH in an Escherichia coli strain lacking the bacterial homolog. Expression of the parasite gene restores growth of this mutant on minimal medium, confirming that the protein has IMPDH activity. The recombinant protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. The mechanism of the C. parvum enzyme involves the random addition of substrates and ordered release of products with rate-limiting hydrolysis of a covalent enzyme intermediate. The pronounced resistance of C. parvum IMPDH to mycophenolic acid inhibition is in strong agreement with its bacterial origin. The values of K-m for NAD and K-i for mycophenolic acid as well as the synergistic interaction between tiazofurin and ADP differ significantly from those of the human enzymes. These data suggest that the structure and dynamic properties of the NAD binding site of C. parvum IMPDH can be exploited to develop parasite-specific inhibitors.