umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Show others and affiliations
2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 51, 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 α2β2complex, 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
American Society for Biochemistry and Molecular Biology, 2008. Vol. 283, no 51, 35310-35318 p.
Keyword [en]
E. coli ribonucleotide reductase, RNR, oligomerization, octamer, ATP, dATP
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
Identifiers
URN: urn:nbn:se:umu:diva-10704DOI: 10.1074/jbc.M806738200PubMedID: 18835811OAI: oai:DiVA.org:umu-10704DiVA: diva2:150375
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2008-10-23 Created: 2008-10-23 Last updated: 2014-01-22Bibliographically approved
In thesis
1. Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei
Open this publication in new window or tab >>Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs.

T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease.

It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice.

The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains.

T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2013. 48 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1524
Keyword
Trypanosoma brucei, adenosine kinase, thymidine kinase, methylthioadenosine phosphorylase, mouser ribonucleotide reductase, E. coli ribonucleotide reductase, RNR, Ara-A, F-Ara-A, dNTP, NTP, doexynucleotide metabolism, nucleosides, nucleoside kinases, allosteric regulation
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-80904 (URN)978-91-7459-737-0 (ISBN)
Public defence
2013-11-08, KB3A9, KBC-huset,, Umeå, 10:00 (English)
Opponent
Supervisors
Projects
Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.
Funder
Swedish Research CouncilSida - Swedish International Development Cooperation Agency
Available from: 2013-10-18 Created: 2013-09-27 Last updated: 2013-10-18Bibliographically approved
2. DNA precursor biosynthesis-allosteric regulation and medical applications
Open this publication in new window or tab >>DNA precursor biosynthesis-allosteric regulation and medical applications
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound.

In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes.

An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness.

Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik, 2008. 29 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1175
Keyword
Biochemistry, DNA biosynthesis, ribonucleotide reductase, allosteric regulation, Trypanosoma brucei, adenosine kinase, nucleoside analogs, mitochondrial DNA, TWINKLE, Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-1678 (URN)978-91-7264-558-5 (ISBN)
Public defence
2008-06-13, KB3A9, KBC Huset, Umeå University, SE-90187, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2014-01-22Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Authority records BETA

Rofougaran, RezaVodnala, MunenderHofer, Anders

Search in DiVA

By author/editor
Rofougaran, RezaCrona, MikaelVodnala, MunenderSjöberg, Britt-MarieHofer, Anders
By organisation
Department of Medical Biochemistry and BiophysicsDepartment of Molecular Biology (Faculty of Medicine)
In the same journal
Journal of Biological Chemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 171 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf