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High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states: an unusual flavodoxin
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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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.

Place, publisher, year, edition, pages
2010. Vol. 277, no 20, 4265-4277 p.
Keyword [en]
crystal structure; flavin mononucleotide; flavodoxin; NrdI; ribonucleotide reductase
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-43637DOI: 10.1111/j.1742-4658.2010.07815.xISI: 000282326600014OAI: oai:DiVA.org:su-43637DiVA: diva2:358741
Note
authorCount :7Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2017-12-12Bibliographically approved
In thesis
1. The evolution of ribonucleotide reductases
Open this publication in new window or tab >>The evolution of ribonucleotide reductases
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Evolutionen av ribonukleotidreduktas
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
Evolution, ribonucleotide reductase, horizontal gene transfer, phylogeny, protein classification
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-43653 (URN)978-91-7447-163-2 (ISBN)
Public defence
2010-12-03, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
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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

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