O'Handley S F, Dunn C A, Bessman M J
Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
J Biol Chem. 2001 Feb 23;276(8):5421-6. doi: 10.1074/jbc.M004100200. Epub 2000 Oct 26.
Orf135 from Escherichia coli is a new member of the Nudix (nucleoside diphosphate linked to some other moiety, x) hydrolase family of enzymes with substrate specificity for CTP, dCTP, and 5-methyl-dCTP. The gene has been cloned for overexpression, and the protein has been overproduced, purified, and characterized. Orf135 is most active on 5-methyl-dCTP (k(cat)/K(m) = 301,000 M(-1) s(-1)), followed by CTP (k(cat)/K(m) = 47,000 M(-1) s(-1)) and dCTP (k(cat)/K(m) = 18,000 M(-1) s(-1)). Unlike other nucleoside triphosphate pyrophophohydrolases of the Nudix hydrolase family discovered thus far, Orf135 is highly specific for pyrimidine (deoxy)nucleoside triphosphates. Like other Nudix hydrolases, the enzyme cleaves its substrates to produce a nucleoside monophosphate and inorganic pyrophosphate, has an alkaline pH optimum, and requires a divalent metal cation for catalysis, with magnesium yielding optimal activity. Because of the nature of its substrate specificity, Orf135 may play a role in pyrimidine biosynthesis, lipid biosynthesis, and in controlling levels of 5-methyl-dCTP in the cell.
来自大肠杆菌的Orf135是Nudix（与其他某个部分相连的核苷二磷酸，x）水解酶家族的新成员，对CTP、dCTP和5-甲基-dCTP具有底物特异性。该基因已被克隆用于过表达，并且该蛋白质已被过量生产、纯化和表征。Orf135对5-甲基-dCTP的活性最高（k(cat)/K(m)=301,000 M(-1)s(-1)），其次是CTP（k(cat)/K(m)=47,000 M(-1)s(-1)）和dCTP（k(cat)/K(m)=18,000 M(-1)s(-1)）。与迄今为止发现的Nudix水解酶家族的其他核苷三磷酸焦磷酸水解酶不同，Orf135对嘧啶（脱氧）核苷三磷酸具有高度特异性。与其他Nudix水解酶一样，该酶切割其底物以产生核苷单磷酸和无机焦磷酸，最适pH为碱性，催化需要二价金属阳离子，镁可产生最佳活性。由于其底物特异性的性质，Orf135可能在嘧啶生物合成、脂质生物合成以及控制细胞中5-甲基-dCTP的水平中发挥作用。
