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 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.
O'Handley
S F
SF
Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA. sohandle@richmond.edu
Dunn
C A
CA
Bessman
M J
MJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D011755:Pyrophosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000096964:Nudix Hydrolases
D003570:Cytidine Triphosphate; D003845:Deoxycytosine Nucleotides; D011755:Pyrophosphatases; D011994:Recombinant Proteins; C024107:2'-deoxycytidine 5'-triphosphate; C034817:5-methyldeoxycytidine triphosphate; C422942:nudix hydrolase Orf135; D000096964:Nudix Hydrolases