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- Crona, Mikael, et al.
(författare)
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NrdH-Redoxin Protein Mediates High Enzyme Activity in Manganese-reconstituted Ribonucleotide Reductase from Bacillus anthracis
- 2011
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Ingår i: Journal of Biological Chemistry. - Bethesda, Md. : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 286:38, s. 33053-33060
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.
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- Crona, Mikael, 1981-, et al.
(författare)
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Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
- 2010
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 23:8, s. 633-641
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors
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- Johansson, Renzo, et al.
(författare)
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High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:20, s. 4265-4277
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Lundin, Daniel, 1965-, et al.
(författare)
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Ribonucleotide reduction : horizontal transfer of a required function spans all three domains
- 2010
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Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 10:383
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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- Lundin, Daniel, 1965-, et al.
(författare)
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RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank
- 2009
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes. PMID: 19995434
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- Nord, David, et al.
(författare)
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A functional homing endonuclease in the Bacillus anthracis nrdE group I intron
- 2007
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 189:14, s. 5293-5301
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Tidskriftsartikel (refereegranskat)abstract
- The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3' extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G + C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing IVSs within the coding region of the nrdE gene.
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- Sjöberg, Britt-Marie, et al.
(författare)
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Shift in Ribonucleotide Reductase Gene Expression in Pseudomonas aeruginosa during Infection
- 2011
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 79:7, s. 2663-2669
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Tidskriftsartikel (refereegranskat)abstract
- The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients.
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