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The evolution of ribonucleotide reductases
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Evolutionen av ribonukleotidreduktas (Swedish)
Abstract [en]

Ribonucleotide reductase (RNR) catalyses the transformation of RNA building blocks, ribonucleotides, to DNA building blocks, deoxyribonucleotides. This is the only extant reaction pathway for de novo synthesis of DNA building blocks and the enzyme is thus necessary for life. RNR is found in all but a few organisms.

There are three classes of RNR, all evolutionarily related. The classification is built on differences in generation of the radical that is central to the reaction. As a consequence, RNR classes have different operational constraints. Class I requires oxygen, while class III is poisoned by oxygen. Class II is independent of oxygen, but dependent on vitamin B12 and, hence, cobalt. This makes RNR interesting from an evolutionary as well as environmental point of view.

RNR must have evolved before the transition from RNA encoded genomes to DNA encoded. The extant radical-based reaction is likely similar to the ancestral reaction, which entails that the ancestral enzyme was a protein and not an RNR.

My results are consistent with both class II and III being present in the last universal common ancestor. Class I RNR evolved later, presumably once oxygen levels had risen. From commonalities in extant RNRs we can reconstruct their last common ancestor as an enzyme that 1) used a transient cysteine-radical in the reaction, 2) reduced all four ribonucleotides, 3) regulated which nucleotide was reduced after binding an effector nucleotide in the dimer interface of the enzyme and 4) had activity regulation through binding of a nucleotide to another part of the enzyme.

The presence of a specific RNR class is likely to influence the environmental range of organisms, which makes horizontal transfer of RNR particularly interesting. Horizontal transfer of RNR genes is widespread, both between closely related organisms and between domains. For instance, all three classes are present in eukaryotes, but likely all three are results of horizontal transfer.

Abstract [sv]

Ribonukleotidreduktas (RNR) katalyserar omvandlingen av RNA-byggstenar, ribonukleotider, till DNA-byggstenar, deoxyribonukleotider. Reaktionsvägen är den enda kända som resulterar i DNA-byggstenar, och RNR är därmed en nödvändigt för alla levande organismer. RNR-gener finns också i stort sett i alla organismer.

Det finns tre klasser av RNR, som alla är evolutionärt besläktade med varandra. Klassindelningen bygger på hur den radikal som är viktig för reaktionen skapas. De olika klasserna är beroende av sin miljö på olika sätt. Klass I kräver syre medan klass III inte tål syre. Klass II är oberoende av syre, men är istället beroende av vitamin B12 och, därmed, kobolt.

RNR måste ha uppstått innan DNA blev den informationsbärande molekylen i organismer. Det ursprungliga RNR-enzymet måste ha varit ett protein, och inte en RNA-molekyl, p.g.a. den fria radikalen i reaktionen.

Enligt mina resultat kan både klass II- och klass III-RNR ha funnits i den gemensamma förfadern till allt cellulärt liv. Klass I-RNR uppstod senare, när atmosfären började innehålla tillräckligt med syre. Utifrån gemensamma egenskaper i dagens RNRer kan vi dra slutsatsen att proteinet som de två klasserna uppstod ur 1) använde en transient cystein-radikal i reaktionen, 2) kunde katalysera reduktionen av alla fyra nukleotider, 3) reglerade vilken som reducerades genom att binda en nukleotid mellan monomererna och 4) att aktiviteten i enzymet reglerades genom bindning av en nukleotid, i en annan del av enzymet.

Förekomsten av en RNR-klass i en organism är troligen en viktig faktor för vilka miljöer den kan trivas i, vilket gör horisontell överföring av RNR-gener extra intressant. Horizontell överföring är vanligt, såväl mellan nära som avlägset besläktade organismer. I eukaryoter t.ex. finns idag alla tre klasser representerade men alla verkar vara resultatet av horisontell överföring.

Place, publisher, year, edition, pages
Stockholm: Department of molecular biology and functional genomics, Stockholm University , 2010. , 59 p.
Keyword [en]
Evolution, ribonucleotide reductase, horizontal gene transfer, phylogeny, protein classification
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-43653ISBN: 978-91-7447-163-2 (print)OAI: oai:DiVA.org:su-43653DiVA: diva2:358856
Public defence
2010-12-03, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper nr. 2: Manuscript; Paper nr 3: Manuscript.Available from: 2010-11-11 Created: 2010-10-25 Last updated: 2010-11-15Bibliographically approved
List of papers
1.
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2. Ribonucleotide reduction: horizontal transfer of a required function spans all three domains
Open this publication in new window or tab >>Ribonucleotide reduction: horizontal transfer of a required function spans all three domains
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2010 (English)In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 10, no 383Article in journal (Other academic) Published
Abstract [en]

Background Ribonucleotide reduction is the only de novo pathway for synthesis ofdeoxyribonucleotides, the building blocks of DNA. The reaction is catalysed byribonucleotide reductases (RNRs), an ancient enzyme family comprised of threeclasses. Each class has distinct operational constraints, and are broadly distributedacross organisms from all three domains, though few class I RNRs have beenidentified in archaeal genomes, and classes II and III likewise appear rare acrosseukaryotes. In this study, we examine whether this distribution is best explained bypresence of all three classes in the Last Universal Common Ancestor (LUCA), or byhorizontal gene transfer (HGT) of RNR genes. We also examine to what extentenvironmental factors may have impacted the distribution of RNR classes.

Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possesseda class I RNR, but that the eukaryotic class I enzymes are not directly descended fromclass I RNRs in archaea. Instead, our results indicate that archaeal class I RNR geneshave been independently transferred from bacteria on two occasions. While LECApossessed a class I RNR, our trees indicate that this is ultimately bacterial in origin.We also find convincing evidence that eukaryotic class I RNR has been transferred tothe bacteroidetes, providing a stunning example of HGT from eukaryotes back tobacteria. Based on our phylogenies and available genetic and genomic evidence, classII and III RNRs in eukaryotes also appear to have been transferred from bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of archaeaand eukaryotes appears, through a process of elimination, to have been a dimeric classII RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT.

Conclusions Horizontal gene transfer has clearly played an important role in the evolution of theRNR repertoire of organisms from all three domains of life. Our results clearly showthat class I RNRs have spread to archaea and eukaryotes via transfers from thebacterial domain, indicating that class I likely evolved in the bacteria. We find noclear evolutionary trace placing either class II or III RNRs in the LUCA, despite thefact that ribonucleotide reduction is an essential cellular reaction and was pivotal tothe transition from RNA to DNA genomes. Instead, a general pattern emerges whereenvironmental and enzyme operational constraints, especially the presence or absenceof oxygen, coupled with horizontal transmission are major determinants of the RNR repertoire of genomes.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-43635 (URN)10.1186/1471-2148-10-383 (DOI)
Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2017-12-12Bibliographically approved
3. The functional diversity and evolutionary relationships of ferritin-like proteins
Open this publication in new window or tab >>The functional diversity and evolutionary relationships of ferritin-like proteins
(English)Manuscript (preprint) (Other academic)
Abstract [en]

BackgroundThe ferritin-like proteins are evolutionarily related, as evidenced by the topology oftheir signature metal-binding four-helix bundle. They perform diverse functions suchas iron/oxygen detoxification, iron storage, DNA protection and substrate oxidation.Though evolutionarily related, sequence similarity between families and often withinfamilies is low. To analyse the evolutionary relationships between individual familiesand their functional roles systematically, we turned to 3D structural alignment andphylogenetic methods.ResultsOur phylogenetic network recovers all characterised functional groups of ferritin-likeproteins and suggests evolutionary relationships between them. The evolutionaryhypotheses that are suggested by the phylogeny are tested against availableindependent evidence such as dimerisation geometries, qualitative comparison ofstructures and sequence based partial phylogenies. Generally our hypotheses stand upagainst the evidence, but in a few cases verification have to await further data.ConclusionsTwo large evolutionary groups of ferritin-like proteins are identified from structuralphylogeny: ferritins, bacterioferritins and Dps proteins on one hand, and substrateoxidising proteins, bacterial multicomponent monooxygenases, fatty acid desaturasesand class I ribonucleotide reductase radical generating subunits, on the other. Themethod we present provides a robust way of evolutionarily classifying a functionallydiverse group of distantly related proteins, as well as examining the possible functionsof poorly-characterised proteins.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-43636 (URN)
Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2012-01-17Bibliographically approved
4. High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states: an unusual flavodoxin
Open this publication in new window or tab >>High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states: an unusual flavodoxin
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2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 20, 4265-4277 p.Article in journal (Refereed) Published
Abstract [en]

The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4-10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

Keyword
crystal structure; flavin mononucleotide; flavodoxin; NrdI; ribonucleotide reductase
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-43637 (URN)10.1111/j.1742-4658.2010.07815.x (DOI)000282326600014 ()
Note
authorCount :7Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2017-12-12Bibliographically approved

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