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Enzymatically active mammalian ribonucleotide reductase exists primarily as an α6β2 octamer
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 38, 27705-27711 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an alpha(2)beta(2) complex consisting of R1 (alpha) and R2 (beta) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (alpha(6)beta(2)). The newly discovered alpha(6)beta(2) complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.

Place, publisher, year, edition, pages
2006. Vol. 281, no 38, 27705-27711 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-22397DOI: 10.1074/jbc.M605573200PubMedID: 16861739OAI: oai:DiVA.org:umu-22397DiVA: diva2:216284
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved
In thesis
1. 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
2. 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

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