Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.
Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).
Eliasson
R
R
Department of Biochemistry, Karolinska Institute, Stockholm, Sweden.
Pontis
E
E
Fontecave
M
M
Gerez
C
C
Harder
J
J
Jörnvall
H
H
Krook
M
M
Reichard
P
P
D000332:Aerobiosis; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D012264:Ribonucleotide Reductases