NADPH-sulfite reductase from Escherichia coli. A flavin reductase participating in the generation of the free radical of ribonucleotide reductase.
Protein R2, the small subunit of ribonucleotide reductase of Escherichia coli, contains an essential free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged by hydroxyurea, the enzyme is transformed into an inactive form, metR2. E. coli contains a NAD(P)H:flavin oxidoreductase, named Fre, absolutely required for the regeneration of the radical and the activation of metR2 into R2. Consequently, an E. coli mutant strain lacking an active fre gene is more sensitive to hydroxyurea during growth, demonstrating the physiological protective function of Fre from the loss of the radical. However, this gene is not essential, and we found that E. coli contains a second tyrosyl radical generating activity, also residing in a flavin reductase. The enzyme has been purified 200-fold to homogeneity and found to be identical to sulfite reductase. Pure sulfite reductase has the ability to catalyze the reduction of free riboflavin, FMN, or FAD by NADPH and thus, as Fre, to transfer electrons to the iron center of metR2, a key step during the activation reaction.
Covès
J
J
Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, Unité de Recherche, Université Joseph Fourier, Grenoble, France.
Nivière
V
V
Eschenbrenner
M
M
Fontecave
M
M
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005609:Free Radicals; D006918:Hydroxyurea / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D050864:Sulfite Reductase (NADPH); D014443:Tyrosine / Q000378:metabolism
D005609:Free Radicals; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D009249:NADP; D005486:Flavin Mononucleotide; D012264:Ribonucleotide Reductases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D050864:Sulfite Reductase (NADPH); C494314:sulfite reductase (NADPH), E coli; D006918:Hydroxyurea