Orf135 from Escherichia coli Is a Nudix hydrolase specific for CTP, dCTP, and 5-methyl-dCTP

J Biol Chem. 2001 Feb 23;276(8):5421-6. doi: 10.1074/jbc.M004100200. Epub 2000 Oct 26.

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Cytidine Triphosphate / metabolism*
  • Deoxycytosine Nucleotides / metabolism*
  • Escherichia coli / enzymology*
  • Molecular Sequence Data
  • Multigene Family
  • Pyrophosphatases / genetics
  • Pyrophosphatases / isolation & purification
  • Pyrophosphatases / metabolism*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Deoxycytosine Nucleotides
  • Recombinant Proteins
  • 2'-deoxycytidine 5'-triphosphate
  • 5-methyldeoxycytidine triphosphate
  • Cytidine Triphosphate
  • Pyrophosphatases
  • nudix hydrolase Orf135