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Class I Ribonucleotide Reductases: overall activity regulation, oligomerization, and drug targeting
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Anders Hofer)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity.  The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR.

Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively.  The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group.

We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 51+8 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1894
Keyword [en]
Ribonucleotide reductase, GEMMA, Allosteric regulation
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-133817ISBN: 978-91-7601-703-6 OAI: oai:DiVA.org:umu-133817DiVA: diva2:1089013
Public defence
2017-05-12, KB.E3.01 Lilla Hörsalen, KBC huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-04-21 Created: 2017-04-18 Last updated: 2017-05-05Bibliographically approved
List of papers
1. Diversity in Overall Activity Regulation of Ribonucleotide Reductase
Open this publication in new window or tab >>Diversity in Overall Activity Regulation of Ribonucleotide Reductase
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 28, 17339-17348 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109543 (URN)10.1074/jbc.M115.649624 (DOI)000357730900029 ()25971975 (PubMedID)
Funder
Swedish Research CouncilThe Kempe FoundationsCarl Tryggers foundation
Available from: 2015-09-30 Created: 2015-09-30 Last updated: 2017-04-18Bibliographically approved
2. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones
Open this publication in new window or tab >>Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones
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2016 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 6, 906-917 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-124103 (URN)10.1016/j.str.2016.03.025 (DOI)000377782200011 ()27133024 (PubMedID)
External cooperation:
Available from: 2016-07-17 Created: 2016-07-17 Last updated: 2017-04-18Bibliographically approved
3. A ribonucleotide reductase inhibitor with deoxyribonucleoside-reversible cytotoxicity
Open this publication in new window or tab >>A ribonucleotide reductase inhibitor with deoxyribonucleoside-reversible cytotoxicity
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2016 (English)In: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261, Vol. 10, no 9, 1375-1386 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL-60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR-targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea. 

Keyword
Ribonucleotide reductase, Nucleotide metabolism, Inhibitors, Antiproliferative compounds, Cell cycle, Cytotoxicity, Oligomeric state, GEMMA
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-129926 (URN)10.1016/j.molonc.2016.07.008 (DOI)000386860800001 ()27511871 (PubMedID)
Available from: 2017-01-10 Created: 2017-01-10 Last updated: 2017-04-18Bibliographically approved

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