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Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R1
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 15, 14997-15003 p.Article in journal (Refereed) Published
Abstract [en]

Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (α2) and R2 (β2), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

Place, publisher, year, edition, pages
2005. Vol. 280, no 15, 14997-15003 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-23548DOI: 10.1074/jbc.M500565200OAI: oai:DiVA.org:su-23548DiVA: diva2:192719
Available from: 2005-01-26 Created: 2005-01-26 Last updated: 2017-12-13Bibliographically approved
In thesis
1. The influence of nucleotides on ribonucleotide reductase assambly in class I ribonucleotide reductase from Escherichia coli
Open this publication in new window or tab >>The influence of nucleotides on ribonucleotide reductase assambly in class I ribonucleotide reductase from Escherichia coli
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The components of DNA, the deoxyribonucleotides, are produced from the components of RNA, the ribonucleotides. One single substitution is needed to convert a ribonucleotide into a deoxyribonucleotide i.e. a replacement of a hydroxyl group with a hydrogen atom. The reaction is catalysed by ribonucleotide reductase, an enzyme that is present in all living organisms. At first this conversion may seem trivial but in reality it is a difficult chemical reaction requiring much energy. In ribonucleotide reductase this energy is provided by an amino acid radical that upon each catalytic turnover is transferred from its stable position in the R2 protein to the active site of protein R1. The need for deoxyribonucleotides in the cell varies, therefore the activity of ribonucleotide reductase must be regulated. A complex allosteric regulation controls both the level of enzymatic activity and the substrate specificity to make sure that the deoxyribonucleotides are produced in correct amounts. In this work, we have shown that the reason why enzymatic activity is turned off when dATP binds is due to formation of a constrained R1: R2 interaction and we have proposed that a conserved hydrogen bond is important in this mechanism. We evaluated the effect of nucleotides on the R1: R2 interaction further using the surface plasmon resonance technique and found that allosteric effectors and substrates as well as the presence of thioredoxin considerably enhances the interaction.

The second allosteric site that controls substrate specificity is located at the interaction area of the two polypeptides constituting protein R1. We have identified key residues in the dimerisation process of the two polypeptides and we have established a stabilisation effect of allosteric effectors and substrates on the dimer interaction using a mutant protein. The radical is believed to be transferred between the two proteins that constitute ribonucleotide reductase by a chain of hydrogen bonded amino acid residues. We have developed a new in vivo activity assay in which we have shown the physiological importance of these residues. Another methodological approach in this work was an attempt to turn protein R1 of ribonucleotide reductase into a selenoprotein by genetically substituting one of the active site cysteines for a selenocysteine. In a selenocysteine-substituted protein, this particular residue can be distinguished from the other cysteines which would be advantageous in subsequent biophysical characterisations of thiyl radicals.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik, 2005. 48 p.
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-343 (URN)91-7265-997-1 (ISBN)
Public defence
2005-02-21, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 09:00
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Available from: 2005-01-26 Created: 2005-01-26Bibliographically approved

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