Abstract
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.
Highlights
The Nudix hydrolases are a family of enzymes catalyzing the hydrolysis of substrates consisting of a nucleoside diphosphate linked to some other moiety, x, and are defined by the signature sequence GX5EX7REUXEEXGU where U is a bulky aliphatic amino acid, Ile, Leu, or Val [1]
Orf135 from Escherichia coli is a new member of the Nudix hydrolase family of enzymes with substrate specificity for CTP, dCTP, and 5-methyl-dCTP
The closest matches to identified enzymes are to MutT from Proteus vulgaris, Haemophilus influenzae, and E. coli with E values of 5 ϫ 10Ϫ10, 3 ϫ 10Ϫ9, and 8 ϫ 10Ϫ8, respectively
Summary
The Nudix hydrolases are a family of enzymes catalyzing the hydrolysis of substrates consisting of a nucleoside diphosphate linked to some other moiety, x (the acronym Nudix), and are defined by the signature sequence GX5EX7REUXEEXGU where U is a bulky aliphatic amino acid, Ile, Leu, or Val [1]. The family has grown to include over 450 open reading frames in over 85 species as well as a variety of enzymes as indicated above. Orf135 is a true member of the Nudix hydrolase family; it contains the signature sequence GX5EX7REUXEEXGU and it cleaves the nucleoside diphosphate derivatives CTP, dCTP, and 5-methyl-dCTP. It is unique in its substrate specificity, because it is the first Nudix hydrolase highly specific toward pyrimidine substrates. We describe the cloning and expression of the orf135 gene and the purification and characterization of the Orf135 enzyme, and we discuss its possible role in intermediary metabolism
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