Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione.
E. coli B tsnC 7004, an E. coli B/1 mutant with normal phenotype unable to replicate phage T7 DNA [Chamberlin, M. (1974)J. Virol. 14,509-516], contained no detectable level of thioredoxin when assayed with ribonucleotide reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Gently lysed E. coli tsnC 7004 cell extracts reduced CDP when supplemented with NADPH as efficiently as the parent strain E. coli B/1 despite the lack of thioredoxin, indicating the presence of another hydrogen transport system. This could be divided into two parts by heat treatment at 85degrees; one heat-stable fraction, which was active in the presence of dithiothreitol or glutathione, and one heat-labile fraction. Addition of yeast glutathione reductase [NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2] to the heated extracts restored full activity. The results demonstrate a novel hydrogen transport system in E. coli consisting of NADPH, glutathione, glutathione reductase, and a heat-stable enzyme called "glutaredoxin". Reduced glutathione at physiological concentrations functions as hydrogen donor for ribonucleotide reduction only in the presence of glutaredoxin. Glutaredoxin was not reduced by E. coli thioredoxin reductase (NADPH:oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) and showed no crossreaction with antibodies against thioredoxin. These results demonstrate the existence of two different electron transfer systems from NADPH to deoxyribonucleotides and provide a function for glutathione in DNA synthesis.
D004229:Dithiothreitol / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism*; D005980:Glutathione Reductase / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D009154:Mutation; D009249:NADP / Q000378:metabolism; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism
D013879:Thioredoxins; D009249:NADP; D006859:Hydrogen; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D005980:Glutathione Reductase; D005978:Glutathione; D004229:Dithiothreitol