Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii.
A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP. Neither endonuclease nor 3'-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.
Chung
J H
JH
Structural Biology Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, Korea and The Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea.
Back
J H
JH
Park
Y I
YI
Han
Y S
YS
D019609:Archaeoglobus / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003850:Deoxyribonuclease I / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019271:Hypoxanthine / Q000378:metabolism*; D007293:Inosine Triphosphate / Q000378:metabolism; D017017:Methanococcus / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D019820:Xanthine / Q000378:metabolism*
D001426:Bacterial Proteins; D009711:Nucleotides; D009841:Oligonucleotides; D007293:Inosine Triphosphate; D019820:Xanthine; D019271:Hypoxanthine; D003850:Deoxyribonuclease I; D011755:Pyrophosphatases