Orf135 from Escherichia coli is a nudix hydrolase specific for CTP, dCTP, and 5-methyl-dCTP

Orf135 from Escherichia coli is a new member of the Nudix (nucleoside dipho sphate linked to some other moiety, x) hydrolase family of enzymes with sub strate specificity for CTP, dCTP, and 5-methyl-dCTP. The gene has been clon ed for overexpression, and the protein has been overproduced, purified, and characterized. Orf135 is most active on 5-methyl-dCTP (k(cat)/K-m = 301,00 0 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 pyropho phohydrolases 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 op timal activity. Because of the nature of its substrate specificity, Orf135 may play a role in pyrimidine biosynthesis, lipid biosynthesis, and in cont rolling levels of 5-methyl-dCTP in the cell.