Mass Spectrometric Determination of the Radical Scission Site in the Anaerobic Ribonucleotide Reductase of Escherichia coli

King, Dan; Reichard, Peter

doi:10.1006/bbrc.1995.1103

Cited by 43 publications

(29 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Given that class Ib RNRs require O 2 for cofactor assembly whereas class III enzymes employ a glycyl radical that causes cleavage of the ␣ subunit (NrdD) upon exposure to O 2 ( Fig. 1B) (11), this was a critical characteristic to study. During strain construction, cell growth was examined in two environments as follows: in an anaerobic chamber and in an atmosphere of 6% O 2 .…”

Section: Analysis Of Mutant Growth In Broth Culture Under Microaerobimentioning

confidence: 99%

“…However, this requirement is catalytic in that once the active cofactor (Fe III 2 -Y ⅐ or Mn III 2 -Y ⅐ ) is generated, it catalyzes many turnovers of nucleotides to deoxynucleotides. However, the cofactor in the class III RNRs (NrdDG) must be assembled under strictly anaerobic conditions as the [4Fe4S] ϩ cluster in NrdG is inactivated by O 2 and the active glycyl radical generated by NrdG and S-adenosylmethionine reacts rapidly with O 2 resulting in cleavage of the NrdD polypeptide chain and permanent enzyme inactivation (11). In class II (NrdJ) enzymes, O 2 is neither required for activity nor detrimental to the enzyme.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Rhodes

Crump

Makhlynets³

et al. 2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

Section: Analysis Of Mutant Growth In Broth Culture Under Microaerobimentioning

confidence: 99%

mentioning

confidence: 99%

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Rhodes

Crump

Makhlynets³

et al. 2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…These enzymes are therefore limited to aerobic conditions. In contrast, the glycyl radical of class III is rapidly destroyed by oxygen, with cleavage of the polypeptide chain at the location of the radical (49). Class III enzymes are therefore strictly anaerobic.…”

Section: Three Classes: An Overviewmentioning

confidence: 99%

Ribonucleotide Reductases

Jordan

Reichard

1998

Annu. Rev. Biochem.

Self Cite

664

639

View full text Add to dashboard Cite

Ribonucleotide reductases provide the building blocks for DNA replication in all living cells. Three different classes of enzymes use protein free radicals to activate the substrate. Aerobic class I enzymes generate a tyrosyl radical with an ironoxygen center and dioxygen, class II enzymes employ adenosylcobalamin, and the anaerobic class III enzymes generate a glycyl radical from S-adenosylmethionine and an iron-sulfur cluster. The X-ray structure of the class I Escherichia coli enzyme, including forms that bind substrate and allosteric effectors, confirms previous models of catalytic and allosteric mechanisms. This structure suggests considerable mobility of the protein during catalysis and, together with experiments involving site-directed mutants, suggests a mechanism for radical transfer from one subunit to the other. Despite large differences between the classes, common catalytic and allosteric mechanisms, as well as retention of critical residues in the protein sequence, suggest a similar tertiary structure and a common origin during evolution. One puzzling aspect is that some organisms contain the genes for several different reductases. CONTENTS

show abstract

“…Like other class III reductases, it is active only under anaerobic conditions and uses nucleoside triphosphates rather than nucleoside diphosphates as substrates. The requirement for anaerobic conditions is believed to be due to the deleterious effect of molecular oxygen, which reacts with the radical harbored at glycine 580 within the NrdD subunit, resulting in cleavage of the polypeptide backbone at or near this residue (14), as has been demonstrated for the corresponding enzyme in E. coli (15,16). Formation of the glycyl radical requires the presence of the NrdG subunit, which contains an iron-sulfur center (16,14).…”

mentioning

confidence: 99%

Localization and Characterization of Two Nucleotide-binding Sites on the Anaerobic Ribonucleotide Reductase from Bacteriophage T4

Olcott

Andersson

Sjöberg

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have used 8-azidoadenosine 5-triphosphate (8-N 3 ATP) to investigate the nucleotide-binding sites on the NrdD subunit of the anaerobic ribonucleotide reductase from T4 phage. Saturation studies revealed two saturable sites for this photoaffinity analog of ATP. One site exhibited half-maximal saturation at approximately 5 M [␥-32 P]8-N 3 ATP, whereas the other site required 45 M. To localize the sites of photoinsertion, photolabeled peptides from tryptic and chymotryptic digests were isolated by immobilized Al 3؉ affinity chromatography and high performance liquid chromatography and subjected to amino acid sequence and mass spectrometric analyses. The molecular masses of the photolabeled products of cyanogen bromide cleavage were estimated using tricine-SDS-polyacrylamide gel electrophoresis. Overlapping sequence analysis localized the higher affinity site to the region corresponding to residues 289 -291 and the other site to the region corresponding to residues 147-160. Site-directed mutagenesis of Cys 290 , a residue conserved in all known class III reductases, resulted in a protein that exhibited less than 10% of wild type enzymatic activity. These observations indicate that Cys 290 may reside in or near the active site. High performance liquid chromatography analysis revealed that photoinsertion of [␥-32 P]8-N 3 ATP into the site corresponding to residues 147-160 was almost completely abolished when 100 M dATP, dGTP, or dTTP was included in the photolabeling reaction mixture, whereas 100 M ATP, GTP, CTP, or dCTP had virtually no effect. Based on these nucleotide binding properties, we conclude that this site is an allosteric site analogous to the one that has been shown to regulate substrate specificity of other ribonucleotide reductases. There was no evidence for a second allosteric nucleotide-binding site as observed in the anaerobic ribonucleotide reductase from Escherichia coli.Ribonucleotide reductases catalyze the reduction of ribonucleotides to their corresponding 2Ј-deoxyribonucleotides. Currently, they are divided into three classes based on differences in cofactor requirements, structural composition, and type of radical employed for catalysis (1, 2). Because of the importance of maintaining a balanced supply of deoxyribonucleotides for DNA synthesis (3, 4), they are enzymes that are subject to complex allosteric regulation (5, 6). Although there may be striking differences in primary sequence, the same general mechanism for allosteric regulation appears to apply to all ribonucleotide reductases described to date, with only subtle differences observed (7). Three different nucleotide-binding sites have been localized on the prototypical class I reductase from Escherichia coli using photoaffinity labeling (8) and x-ray crystallography (9, 10). Two of these sites are allosteric sites that coordinate the reduction of all four ribonucleotide substrates at a single active site. One allosteric site binds only ATP and dATP and regulates the overall activity of the enzyme. The other allosteric site, via i...

show abstract

Mass Spectrometric Determination of the Radical Scission Site in the Anaerobic Ribonucleotide Reductase of Escherichia coli

Cited by 43 publications

References 0 publications

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Ribonucleotide Reductases

Localization and Characterization of Two Nucleotide-binding Sites on the Anaerobic Ribonucleotide Reductase from Bacteriophage T4

Contact Info

Product

Resources

About