Malarial dihydroorotate dehydrogenase
Malarial dihydroorotate dehydrogenase
复制标题
DOI：
10.1074/jbc.m206854200
复制
发表时间：
2002-11-01
影响因子：
4.8
通讯作者：
Phillips, MA
中科院分区：
文献类型：
--
作者：
Baldwin, J;Farajallah, AM;Phillips, MA
疟疾寄生虫依赖于从头嘧啶生物合成来维持其嘧啶库，并且与人类宿主细胞不同，它不能重新合成预先形成的嘧啶。二氢乳清酸脱氢酶（DHODH）催化二氢乳清酸（DHO）氧化生成乳清酸，这是嘧啶生物合成的关键步骤。这种酶位于疟原虫线粒体的外膜上。为了表征疟疾酶的生物化学性质，恶性疟原虫DHODH的N-末端截短形式已在大肠杆菌中表达为可溶性活性酶。杆菌该重组酶结合0.9摩尔当量的辅因子FMN，其pH最大值为8.0（k（cat）8 s（-1），K-m（aPP）DHO（40-80 μ m））。还测定了反应第二步中FMN氧化所需的泛醌辅因子（CoQ（n））的底物特异性。CoQ（n）的类异戊二烯（n）长度是反应效率的决定因素; CoQ（4）、CoQ（6）和癸基泛醌（COQ（D））在反应中被有效利用，然而缺乏类异戊二烯尾部的辅因子（CoQ（0）和维生素K-3）显示出由于K-m（app）增加4至7倍而导致的催化效率降低。五种有效的哺乳动物DHODH抑制剂，氧化还原醛，二氯烯丙基指甲花醌（DCL），和A77 1726的三个类似物作为疟疾酶的抑制剂进行了测试。所有五种化合物都是疟疾酶的不良抑制剂，IC 50范围为0.1-1.0 mm。疟疾酶抑制的IC 50值比哺乳动物酶报道的值高10（2）-10（4）倍，表明抑制剂与DHODH的结合具有物种特异性。这些研究提供了疟疾DHODH活性位点与宿主酶不同的直接证据，并且它是开发新的抗疟疾剂的有吸引力的靶标。
The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate, (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K-m(aPP) DHO (40-80 mum)). The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (COQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K-3) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K-m(app). Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC50's ranging from 0.1-1.0 mm. The IC50 values for inhibition of the malarial enzyme are 10(2)-10(4)-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.