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Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Britt-Marie Sjöberg)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Deoxyribonucleic acid (DNA) chains in which our genetic blueprint is stored are built from four DNA precursors by DNA polymerases. The enzyme ribonucleotide reductase (RNR) provides the only de novo synthesis pathway of deoxyribonucleotides from ribonucleotides and is essential for nearly all organisms. All four ribonucleotides are substrates for RNR and key to this flexibility is a sophisticated allosteric regulation. Nucleotide effectors (ATP, dATP, dTTP or dGTP) binding to the allosteric specificity site determines substrate specificity for the active site. When present at high concentrations, dATP binds to the allosteric overall activity site and inhibits activity by an unknown mechanism. Three approaches, RNR activity measurements, subunit interaction studies and quaternary structure studies were applied to four different class I RNRs to address the allosteric overall regulation. We found that allosteric overall inhibition was closely linked to formation of tight and large RNR protein complexes; α4β4 complex for the Escherichia coli class Ia RNR and α6β2 for the Dictyostelium discoideum class Ia RNR with functional allosteric inhibitions. The Aeh1 phage class Ia RNR with a non-functional dATP inhibition showed weak remnant inhibition features, while the Bacillus anthracis class Ib RNR without the allosteric overall regulation domain lacked these features. In addition, we presented the first biochemical characterization of a mechanism to restore protein function after gene fragmentation, we showed that the B. anthracis class Ib RNR was most active when reconstituted with manganese and in the presence of a physiological redoxin protein and we found that the class Ia RNR is the principal RNR in D. discoideum, although the coexisting class II RNR could partly compensate class I RNR inhibition during axenic growth. Finally, our improved method for studying RNR interactions has potential for RNR inhibitor screening.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University , 2010. , 57 p.
Keyword [en]
Ribonucleotide reductase, allosteric regulation, quaternary structure, subunit interactions, enzyme activity, SPR, GEMMA
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-45740ISBN: 978-91-7447-186-1 (print)OAI: oai:DiVA.org:su-45740DiVA: diva2:369466
Public defence
2010-12-16, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2010-11-24 Created: 2010-11-10 Last updated: 2010-12-06Bibliographically approved
List of papers
1. Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
Open this publication in new window or tab >>Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, 35310-35318 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (α) and R2 (β). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited α4β4 complex in the presence of dATP and an active α2β2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30–50 times stronger in the α4β4 complex than in the α2β2 complex, which was in equilibrium with free α2 and β2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form α4β4 complexes. ATP could also induce the formation of a generally inhibited α4β4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for α4β4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive α6β2 complexes

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology, 2008
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45734 (URN)10.1074/jbc.M806738200 (DOI)000261687900002 ()
Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
2. Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
Open this publication in new window or tab >>Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
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2010 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 23, no 8, 633-641 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors

Place, publisher, year, edition, pages
Oxford University Press, 2010
Keyword
Bacillus anthracis, biotin-streptavidin immobilization, inhibitors, interaction constants, nucleotides
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45735 (URN)10.1093/protein/gzq035 (DOI)000280316000005 ()
Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
3. Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element
Open this publication in new window or tab >>Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 4, 1381-1389 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)2 large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

Place, publisher, year, edition, pages
Oxford University Press, 2011
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45737 (URN)10.1093/nar/gkq924 (DOI)000288019400025 ()
Note
authorCount :6Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
4. NrdH-redoxin mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from Bacillus anthracis
Open this publication in new window or tab >>NrdH-redoxin mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from Bacillus anthracis
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45738 (URN)
Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2010-12-06Bibliographically approved
5. The two classes of ribonucleotide reductase in the social amoeba Dictystelium discoideum
Open this publication in new window or tab >>The two classes of ribonucleotide reductase in the social amoeba Dictystelium discoideum
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45739 (URN)
Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2010-12-06Bibliographically approved

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