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- Aurelius, Oskar, et al.
(författare)
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The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.
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| 2. |
- Crona, Mikael, et al.
(författare)
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A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:12, s. 8198-8208
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.
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| 3. |
- Fagerqvist, Therese, et al.
(författare)
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Off-pathway α-synuclein oligomers seem to alter α-synuclein turnover in a cell model but lack seeding capability in vivo
- 2013
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Ingår i: Amyloid. - : Informa Healthcare. - 1350-6129 .- 1744-2818. ; 20:4, s. 233-244
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Tidskriftsartikel (refereegranskat)abstract
- Aggregated alpha-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Experimental evidence indicates that alpha-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of alpha-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when alpha-synuclein oligomers were added to monomeric alpha-synuclein. In contrast, exogenously added alpha-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing alpha-synuclein that were treated with the oligomers displayed reduced alpha-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected alpha-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of alpha-synuclein oligomers into the neocortex of alpha-synuclein transgenic mice did not induce formation of proteinase K resistant alpha-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced alpha-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.
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| 4. |
- Sahlin, Johan, 1972-
(författare)
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Om kyrklundheten : Värde, kunskap och skrivande i Willy Kyrklunds Om godheten
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The quest for certainty is a constant theme in the works of Swedish-Finnish author Willy Kyrklund. This quest is communicated both explicitly in essayistic prose articles and implicitly as a theme in his works of fiction. The late essay hybrid Om godheten (”On Goodness”, 1988) is a key text in Kyrklund’s oeuvre in this regard as it consists of a mediation between these strategies of communication, the philosophical and the literary. With Om godheten as a starting-point it is the purpose of this study to bring the problem of knowledge to the fore by acknowledging its importance for Kyrklund’s strategies of writing and for his treatment of philosophical questions regarding for example morals and identity. The purpose is also to identify Kyrklund’s use of literature as a cognitive tool with which the reader is lead to reflect on the premises for knowledge and to view the act of reading as parallel to the constructing of knowledge. Furthermore the purpose is to acknowledge the importance of analytical philosophy and natural science in this literary strategy. I consider Finnish philosopher Eino Kaila’s version of logical empiricism an important source of influence for Kyrklund. The gap between empirical (the appeal to experience) and rationalistic (the attempt to generalize) strategies in the strive for knowledge is pointed out as the root problem for logical empiricism by Kaila and this problem has also been touched upon explicitly by Kyrklund. While Kyrklund seems to subscribe to general empiricistic or logical-empiricistic virtues such as empiristic scrutiny, the appeal to science and the rejection of metaphysics, his work does not, however, offer any solutions of how to overcome this gap in any specific logical-empiricistic way. I recognize this gap, as well as the parallel gap between a subjective and an objective standpoint, as a figure of thought in Kyrklund’s literary thinking. Examined in three thematic chapters concerning value, knowledge and writing respectively, the mediation between empiristic and rationalistic tendencies can be found on different levels in Om godheten: in evolutionary approaches to morals versus the teleological approach to morals, in descriptive meta-ethical naturalism versus normative naturalistic ethics, in observations versus laws of nature, in individual expression versus common language, in instances versus generalizing examples and in explorative essayistic modes of writing versus a sovereign metafictive authoral position, to name a few. In putting together traditional text types, genres and modes of writing in new contexts of thought Kyrklund enables a certain way of seeing: combined with several intertextual relations the literary process thus stands out as a parallel to the cognitive process and the epistemological inferiority, where no fundament for certain knowledge is possible, is communicated as an existential problem.
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