Activation of class III ribonucleotide reductase by thioredoxin.
Anaerobic ribonucleotide reductase provides facultative and obligate anaerobic microorganisms with the deoxyribonucleoside triphosphates used for DNA chain elongation and repair. In Escherichia coli, the dimeric alpha2 enzyme contains, in its active form, a glycyl radical essential for the reduction of the substrate. The introduction of the glycyl radical results from the reductive cleavage of S-adenosylmethionine catalyzed by the reduced (4Fe-4S) center of a small activating protein called beta. This activation reaction has long been known to have an absolute requirement for dithiothreitol. Here, we report that thioredoxin, along with NADPH and NADPH:thioredoxin oxidoreductase, efficiently replaces dithiothreitol and reduces an unsuspected critical disulfide bond probably located on the C terminus of the alpha protein. Activation of reduced alpha protein does not require dithiothreitol or thioredoxin anymore, and activation rates are much faster than previously reported. Thus, in E. coli, thioredoxin has very different roles for class I ribonucleotide reductase where it is required for the substrate turnover and class III ribonucleotide reductase where it acts only for the activation of the enzyme.
Padovani
D
D
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, Département de Biologie Moléculaire et Structurale-Chimie Biologie Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, Grenoble, France.
Mulliez
E
E
Fontecave
M
M
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals; D009249:NADP / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013053:Spectrophotometry; D013879:Thioredoxins / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors; D014466:Ultraviolet Rays
D004220:Disulfides; D005609:Free Radicals; D013879:Thioredoxins; D009249:NADP; D012436:S-Adenosylmethionine; D012264:Ribonucleotide Reductases; D003545:Cysteine; D004229:Dithiothreitol