The role and source of 5'-deoxyadenosyl radical in a carbon skeleton rearrangement catalyzed by a plant enzyme.

The last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine. The reaction is catalyzed by a cell-free extract prepared from cultured hairy roots of Datura stramonium. Adenosylmethionine stimulated the rearrangement 10-20-fold and showed saturation kinetics with an apparent Km of 25 microM. It is proposed that S-adenosylmethionine is the source of a 5'-deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B12-dependent enzymes. Possible roles of S-adenosylmethionine as a radical source in higher plants are discussed.

Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction.

The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha2beta2 heterotetramer. In its active form, the large alpha2 subunit contains an oxygen-sensitive glycyl radical, whereas the beta2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to beta2 with a Kd of 10 microM and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta2 and, in the case of alpha2beta2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of beta2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425-11434).

The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center.

During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.

The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681.

The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.

Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

The mechanism of the anaerobic Escherichia coli ribonucleotide reductase investigated with nuclear magnetic resonance spectroscopy.

During the reduction of ribonucleotides with [3H]formate by the class III anaerobic ribonucleotide reductase from Escherichia coli tritium appears in water and not in the product deoxyribonucleotide. In D2O, deuterium replaces the OH-group at carbon-2' with retention of configuration. In addition we find 1-2% deuterium in the 3'-position demonstrating a small exchange of this hydrogen with the protons of water during catalysis. Class I and II enzymes catalyze identical reactions. Members of the three classes of reductases apparently use the same chemical mechanism in spite of having completely different protein structures.