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PMID:8527927
| Citation |
Schellenberger, U, Livi, LL and Santi, DV (1995) Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1. Protein Expr. Purif. 6:423-30 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/prep.1995.1057 |
| Keywords |
Bacillus Phages/enzymology; Bacillus Phages/genetics; Bacillus subtilis/virology; Base Sequence; Cloning, Molecular; Crystallization; Deoxyuracil Nucleotides/metabolism; Escherichia coli/genetics; Gene Expression; Genes, Viral; Genetic Vectors; Hydroxymethyl and Formyl Transferases; Kinetics; Molecular Sequence Data; Molecular Weight; Plasmids/genetics; Protein Conformation; Pyridoxal Phosphate/metabolism; Tetrahydrofolates/metabolism; Transferases/genetics; Transferases/isolation & purification; Transferases/metabolism; Tritium |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0016742: hydroxymethyl-, formyl- and related transferase activity |
ECO:0000314: |
F |
Table 2 shows that deoxyuridylate hydroxymethylase catalyzes the same reaction as L. Casei TS using reactants such as CH2H4 folate and dUMP as well as other reactants in order to yield hmdUMP and H4 Folate. |
complete | ||||
Notes
See also
References
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