Generation of the Glycyl Radical of the Anaerobic Escherichia coli Ribonucleotide Reductase Requires a Specific Activating Enzyme

Sun, Xueyin; Eliasson, R.; Pontis, Elisabet; Andersson, J.; Buist, Girbe; Sjöberg, Britt‐Marie; Reichard, Peter

doi:10.1074/jbc.270.6.2443

Cited by 85 publications

(94 citation statements)

References 18 publications

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“…A close analog of PFL activase is the activating protein of anaerobic ribonucleotide reductase, which also uses AdoMet and reduced flavodoxin for generating a protein-based glycyl radical (11,23,24). Its 4Fe-4S center (10) has a spectroscopic signature (UV-visible and EPR) resembling closely the signature that we have found here for PFL activase.…”

Section: Discussionsupporting

confidence: 53%

Reconstitution and Characterization of the Polynuclear Iron-Sulfur Cluster in Pyruvate Formate-lyase-activating Enzyme

Külzer¹,

Pils²,

Kappl³

et al. 1998

Journal of Biological Chemistry

143

124

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The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys 3Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S] ؉ ) from axial to rhombic symmetry.Pyruvate formate-lyase (PFL) 1 is a key enzyme of the anaerobic glucose fermentation in Escherichia coli and other microorganisms, catalyzing the CoA-dependent cleavage of pyruvate to acetyl-CoA and formate (1). In its active form, this enzyme contains a stable glycyl radical (Gly-734) required for catalysis (2). Studies of mutants and substrate analogs propose that on substrate binding, the spin is transferred from Gly-734 to the reaction center (Cys-418/Cys-419), where a thiyl radical initiates the homolytic cleavage of the pyruvate C-C bond (3, 4).The PFL radical is produced post-translationally by abstraction of the H si atom from the Gly-734 methylene group (5). This occurs by the action of pyruvate formate-lyase-activating enzyme (PFL activase), which employs adenosylmethionine (AdoMet) and dihydroflavodoxin (or artificial 1eϪ donors) as co-substrates, yielding 5Ј-deoxyadenosine and methionine as stoichiometric co-products (Equation 1).Because the abstracted H atom is recovered in the 5Ј-CH 3 of deoxyadenosine, a 5Ј-deoxyadenosyl radical intermediate has been proposed as the actual H abstracting species in this system. How this nucleoside radical is generated from AdoMet is an intriguing problem, requiring in the first place PFL activase to be fully characterized at the molecular level. Previous work has identified PFL activase as a monomer of 28 kDa, and its primary structure, as deduced from the DNAnucleotide sequence (246 amino acids), has been established by N-and C-terminal amino acid sequencing (6, 7). The enzyme has long since been recognized to require Fe 2ϩ for catalytic activity (6), and iron contents in the order of one iron/polypeptide chain were determined in purified prepara...

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Section: Discussionsupporting

confidence: 53%

Reconstitution and Characterization of the Polynuclear Iron-Sulfur Cluster in Pyruvate Formate-lyase-activating Enzyme

Külzer¹,

Pils²,

Kappl³

et al. 1998

Journal of Biological Chemistry

143

124

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“…To substitute cysteine residues in protein ␣ with alanine, the QuikChange mutagenesis protocol (Stratagene) was used with appropriate primers (Table 1) according to the manufacturer. Mutagenesis was carried out on plasmid pRSS, carrying the nrdD gene as a template (12). Mutations were confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase

Logan

Mulliez

Larsson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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A Zn(Cys)4 center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4. The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Mutant enzymes of the homologous RNR from Escherichia coli, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical. The possible roles of the metal center are discussed in relationship to the currently proposed reaction mechanism for generation of the glycyl radical in class III RNRs. Ribonucleotide reductases (RNR) are critical enzymes for the de novo synthesis of DNA in all organisms (1). They catalyze the reduction of the C2ЈOOH bond in ribonucleotides, thereby transforming them to deoxyribonucleotides. Three classes of RNRs have been identified so far that use similar radical chemistry initiated by a diverse range of metallocofactors (2). Class III RNRs are used by facultative anaerobic bacteria such as Escherichia coli and phages such as bacteriophage T4 for their anaerobic growth (3). A class III RNR is an oxygen-sensitive homodimer ␣ 2 , encoded by the nrdD gene, that contains, in its active form, a catalytically essential glycyl radical located on a conserved glycine residue of the C-terminal region of the polypeptide (Gly-681 and Gly-580 in E. coli and T4, respectively).The crystal structure of the oxygen-stable Gly-580 3 Ala mutant class III RNR (nrdD) from bacteriophage T4 has shown that the glycine site is located at the tip of a loop near the C terminus of the polypeptide, Ϸ5 Å away from an essential cysteine residue (Cys-290 in T4) in the substrate-binding site (4, 5). The radical is thus proposed to react with this cysteine to generate an intermediate thiyl radical that serves in turn for abstraction of the H3Ј atom of the ribonucleotide substrate (3, 6). The substrate radical is then reduced to the deoxy form by three reducing equivalents provided by formate and the glycine residue, which is converted back to its radical form for another cycle. No crystal structure of the E. coli enzyme is available yet, but the sequence has 57% identity to that of the RNR from T4. Coupled with the complete conservation of all residues hitherto shown to be critical for reactivity, this similarity allows us to infer mechanistic interpretations for one of the enzymes from structural and functional studies on the other.The activation of protein ␣, i.e., the introduction of the glycyl radical, is the subject of intense studies in our laboratories (7,8). In the enzyme from E. coli, the reaction is mediated by the concerted action of three components: (i) S-adenosylmethionine (AdoMet), which is reduced and cleaved into methionine and a putative 5Ј-deoxyadenosyl radical (Ado°); Ado°then reacts with the glycine residue to generate the glycyl radical by selective hydrogen atom abst...

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“…During preparation of cell-free extract and in assays under air, part of the inactive form could persist and be converted to the active form after restoring reducing conditions. This would resemble the inactivation/activation transition of pyruvate formate lyase or of anaerobic ribonucleotide reductase in Escherichia coli (Knappe and Sawers 1990;Sun et al 1995).…”

Section: Influence Of Added Fumarate and Other Carboxylic Acids On M-mentioning

confidence: 99%

Anaerobic degradation of m -cresol by Desulfobacterium cetonicum is initiated by formation of 3-hydroxybenzylsuccinate

Müller¹,

Galushko²,

Kappler³

et al. 1999

Archives of Microbiology

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The anaerobic bacterium Desulfobacterium cetonicum oxidized m-cresol completely with sulfate as electron acceptor. During growth, 3-hydroxybenzylsuccinate (identified by gas chromatography/mass spectroscopy and by comparison of high-performance liquid chromatography retention time and UV spectrum with a chemically synthesized reference compound) accumulated in the medium. This finding indicates that the methyl group of mcresol is activated by addition to fumarate as in the case of anaerobic toluene metabolism. In cell-free extracts of D. cetonicum, the formation of 3-hydroxybenzylsuccinate from m-cresol and fumarate was detected at an activity of 0.5 nmol min -1 (mg protein) -1 . This reaction depended strictly on anoxic assay conditions. Treatment with air resulted in a complete loss of activity; however, some activity could be recovered after restoring anoxic conditions. The activity was slightly membrane-associated. 3-Hydroxybenzylsuccinate was degraded via CoA thioesterification and further oxidation to 3-hydroxybenzoyl-CoA as subsequent steps in the degradation pathway.

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Generation of the Glycyl Radical of the Anaerobic Escherichia coli Ribonucleotide Reductase Requires a Specific Activating Enzyme

Cited by 85 publications

References 18 publications

Reconstitution and Characterization of the Polynuclear Iron-Sulfur Cluster in Pyruvate Formate-lyase-activating Enzyme

Reconstitution and Characterization of the Polynuclear Iron-Sulfur Cluster in Pyruvate Formate-lyase-activating Enzyme

A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase

Anaerobic degradation of m -cresol by Desulfobacterium cetonicum is initiated by formation of 3-hydroxybenzylsuccinate

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