| 1. |
- Andersson, Jessica, 1967-
(författare)
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Activation, reaction mechanism and allosteric regulation of the anaerobic ribonucleotide reductase from bacteriophage T4
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductase (RNR) catalyse the conversion of ribonucleotides to their corresponding deoxyribonucleotides in all organisms. The deoxyribonucleotides are the building blocks for DNA. Three different classes of RNR are found, class I, II and III. The class I RNRs operate under aerobic conditions, the class III RNRs operate under anaerobic conditions and the class II RNRs are indifferent to oxygen. All classes of RNR catalyse the reaction using a free radical mechanism. The free radical is generated to initiate the reaction mechanism but the generation differs between the classes.I have worked with the anaerobic class III RNR from bacteriophage T4 and the work presented in this thesis involves several different aspects of the enzyme. The class III RNR from phage T4 can be used as a model for other class III RNRs.From isotope labelling experiments, we show that a stable glycyl radical forms in the phage T4 class III RNR. I used site-directed mutagenesis to locate the glycyl radical to Gly580 in the NrdD protein of the T4 class III RNR. The glycyl radical is absolutely required for enzymatic activity.Also using protein engineering, I show for the first time, the importance of cysteines in radical generation and the reaction mechanism of the class III RNRs. Four cysteines in the C-terminal of T4 NrdD are responsible for the last step in the generation of the glycyl radical at Gly580. Two cysteines in the active site of T4 NrdD, Cys79 and Cys290 are required for the reaction mechanism of the enzyme. A third residue within the active site, Asn311 is most likely also important for catalytic activity. A reaction mechanism that is different from the class I and II RNRs has been proposed.The first crystal structure of a class III RNR, the class III RNR from phage T4 is presented. Structural relationships with the known class I RNR structure is discussed as well as similarities with another glycyl-radical enzyme.Finally, the allosteric regulation of the class III RNR from phage T4 was characterized. Almost all RNRs are allosterically regulated to keep the deoxynucleotide pools balanced in the cell. Similarities to other RNRs as well as a unique feature of the class III RNR from phage T4 is discussed.
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| 2. |
- Törnroth, Susanna, 1973-
(författare)
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Structural studies on aerobic and anaerobic respiratory complexes
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All respiratory pathways, whether aerobic or anaerobic, are based on formation of an electrochemical proton gradient called proton motive force (pmf) that drives ATP formation. Membrane-bound respiratory complexes translocate protons across the membrane from a region of low [H+] and negative electrical potential to a region of high [H+] and positive electrical potential. This establishes a proton gradient and a membrane potential that together form pmf. The crystal structures of the respiratory complexes succinate:ubiquinone oxidoreductase (SQR) and formate dehydrogenase-N (Fdh-N) from E. coli, have been solved to 2.6 and 1.6 Å respectively. The structures reveal detailed information about the structure/function relationship of each enzyme as well as demonstrating how pmf is generated. The aerobic respiratory complex SQR is a member of both the citric acid cycle and the respiratory chain. It oxidises succinate to fumarate in the cytoplasm and reduces ubiquinone in the membrane. SQR’s contribution to pmf is by reduction of ubiquinone to ubiquinol, which is subsequently used by other members of the respiratory chain to perform proton translocation. The structure of SQR reveals the location and properties of the ubiquinone binding site and helps explain why mutations in this region result in known diseases in higher organisms.Fdh-N, a member of a major anaerobic respiratory pathway in E. coli, forms a redox loop with dissimilatory nitrate reductase, linked by the menaquinone/menaquinol pool. This redox loop contributes to pmf by translocating two protons from the negative to the positive side and two electrons in the opposite direction. The structure of Fdh-N shows the molecular basis of this process and allows for the proposal of a quinone reduction mechanism.
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| 3. |
- Pankratova, Galina, et al.
(författare)
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Extracellular Electron Transfer by the Gram-positive Bacterium Enterococcus faecalis
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57, s. 4597-4603
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells it is demonstrated that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme-proteins are not involved and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such an intercellular electron transfer might confer syntrophic metabolism that promote growth and other activities of bacteria in the microbiota of humans and animals.
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| 4. |
- Geisler, Daniela, et al.
(författare)
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Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana.
- 2007
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:39, s. 28455-28464
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Tidskriftsartikel (refereegranskat)abstract
- Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca2+ binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca2+, and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca2+ binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca2+ stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca2+-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca2+. The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.
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| 5. |
- Aevarsson, A, et al.
(författare)
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Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase
- 1988
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 232:2, s. 298-302
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Tidskriftsartikel (refereegranskat)abstract
- Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtillis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.
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| 6. |
- Bengtsson, Jenny, et al.
(författare)
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CtaG is required for formation of active cytochrome C oxidase in Bacillus subtilis
- 2004
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Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 150, s. 415-425
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa(3), which is a quinol oxidase, and cytochrome caa(3), which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, Cu-A. B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CrtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p, of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa(3) but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa(3). B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the Cu-A centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.
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| 7. |
- Carlsson Möller, Mirja, et al.
(författare)
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Extracytoplasmatic processes impaired by inactivation of the trxA (thioredoxin gene) in Bacillus subtilis
- 2008
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Ingår i: Journal of Bacteriology. - 0021-9193. ; 190:13, s. 4660-4665
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Tidskriftsartikel (refereegranskat)abstract
- The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.
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| 8. |
- Carlsson, Peter, et al.
(författare)
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Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli
- 1987
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Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 61:2, s. 217-224
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Tidskriftsartikel (refereegranskat)abstract
- The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3). Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively. A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B. subtilis DNA in Escherichia coli. Functional E2o was expressed from the cloned DNA both in B. subtilis and E. coli. E2o had an apparent Mr of 60000 when expressed in E. coli. The B. subtilis E2o component complemented an E. coli E2o-defective mutant in vivo and in vitro. It is concluded that functional B. subtilis E2o can be produced in E. coli and can interact with E. coli and E1o and E3 to form an active chimeric enzyme complex.
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| 9. |
- Carlsson, P., et al.
(författare)
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Genetic Characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively
- 1989
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 171:7, s. 3667-3672
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Tidskriftsartikel (refereegranskat)abstract
- The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.
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| 10. |
- Carlsson, Peter, et al.
(författare)
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In vitro complementation of Bacillus subtilis and Escherichia coli 2-oxoglutarate dehydrogenase complex mutants and genetic mapping of B. subtilis citK and citM mutations
- 1986
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 37:3, s. 373-378
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Tidskriftsartikel (refereegranskat)abstract
- AbstractThe 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (TCA) consists of multiple copies of 3 different subenzymes; E1, E2 and E3. The E3 subenzyme is also a component of the pyruvate dehydrogenase complex. Bacillus subtilis 2-oxoglutarate dehydrogenase mutants were studied. The mutants defective in E1, E2 and E3 subenzyme activity, respectively, could be separated into 3 groups by biochemical complementation analyses. The groups correspond to the citK, citM and citL genes. A B. subtilis subenzyme defect, probably E1, could be complemented with the corresponding Escherichia coli wild-type subenzyme and vice versa. Mutations in citK and citM are closely linked. The gene order kauA--- ---citK-citM was determined from 3-factor transformation crosses. It is concluded that the gene organization and the subenzyme structure of the 2-oxoglutarate dehydrogenase complex are similar in B. subtilis and E. coli.
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