Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1.
2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of [5-3H]dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange.
Schellenberger
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Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Livi
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Santi
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D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics*; D001412:Bacillus subtilis / Q000821:virology; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism; D014166:Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014316:Tritium
D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D014316:Tritium; D011732:Pyridoxal Phosphate; C007267:2'-deoxyuridylic acid; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; C023116:dUMP-hydroxymethylase