Cloning, Expression, Purification, and Characterization of 2′-Deoxyuridylate Hydroxymethylase from Phage SPO1

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 (CH 2H 4folate)-independent tritium exchange of [5- 3H]dUMP for protons of water and dehalogenation of 5-bromo-2′-deoxyuridine-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 CH 2H 4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect an the catalysis of cofactor-independent tritium exchange.