The oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP, is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein.
The Escherichia coli orf135 gene encodes a 15.4kDa protein with homology to the MutT family of nucleotide hydrolases. The orf135 gene was cloned within a glutathione S-transferase (GST) fusion protein expression vector, which was used to overproduce the GST-Orf135 fusion protein in E. coli. The fusion protein thus obtained was purified by affinity column chromatography and gel filtration chromatography from the crude extract. The recombinant Orf135 protein was obtained by removing the GST tag from the purified fusion protein. Various oxidized nucleotides were tested as substrates for the recombinant Orf135 protein. As a result, we found a novel 5-hydroxy-CTPase activity of Orf135, but the hydrolyzing activities for the other nucleotides, including 5-hydroxy-dCTP, were very low. The activation constant (K(a)) of Mg(2+) for the 5-hydroxy-CTPase activity was 1.2 mM, and the pH optimum was 8.5. The catalytic efficiency (k(cat)/K(m)) for this activity was 630 s(-1) mM(-1) at 30 degrees C, which was 30-fold higher than that for the CTPase activity. This result indicates that 5-hydroxy-CTP is the best substrate of Orf135 among the nucleotides tested.
Fujikawa
Katsuyoshi
K
Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
Kasai
Hiroshi
H
D003570:Cytidine Triphosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000096964:Nudix Hydrolases
D003570:Cytidine Triphosphate; D005982:Glutathione Transferase; D011755:Pyrophosphatases; D011993:Recombinant Fusion Proteins; C422942:nudix hydrolase Orf135; D000096964:Nudix Hydrolases