Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.
Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.
Mann
G J
GJ
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Gräslund
A
A
Ochiai
E
E
Ingemarson
R
R
Thelander
L
L
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases