| 1. |
- Hao, Meng-Shu, et al.
(författare)
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The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:9
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Tidskriftsartikel (refereegranskat)abstract
- NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K-0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O-2 and decylubiquinone as electron acceptors. The K-0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K-0.5(Ca2+) for NADPH oxidation was high (approximate to 100 mu M), yet 20-fold lower K-0.5(Ca2+) values were determined at pH 6.8. Lower K-0.5(Ca2+) values were observed with decylubiquinone than with O-2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.
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| 2. |
- Glab, Bartosz, et al.
(författare)
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Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS
- 2016
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 291:48, s. 25066-25076
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Tidskriftsartikel (refereegranskat)abstract
- Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).
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| 3. |
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| 4. |
- Hao, Mengshu
(författare)
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Plant type II NAD(P)H dehydrogenases : Structure, regulation and evolution of NDB proteins
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In living organisms, respiration is a biological process degrading different carbon substrates, consuming O2, and releasing the carbon as CO2. Plants have several alternative enzymes that are involved in the respiratory processes, as compared to animals. These alternative respiratory enzymes allow electrons to be transferred to oxygen in the mitochondrial inner membrane, but bypassing ATP synthesis. The alternative enzymes, e.g., type II NAD(P)H dehydrogenases (NDH-2), affect cellular NAD(P)H redox status, which is of vital importance for energy metabolism, ROS production and removal, anti-oxidation and reductive biosynthesis. Plant NDB-type proteins are NDH-2 enzymes located at the external mitochondrial inner membrane. It was earlier found that NDB1 oxidise cytosolic NADPH, and NDB2 oxidise cytosolic NADH. In this study, the regulatory mechanisms of Arabidopsis thaliana and Solanum tuberosum NDB1 by cytosolic Ca2+ and pH were studied and compared to NDB2, using purified mitochondria and E. coli-produced proteins in a membrane-bound and a purified soluble state. Membrane bound NDB1 and NDB2 oxidised NADPH and NADH, respectively. Soluble forms of NDB1 oxidise both NADH and NADPH, with higher NADPH activity. Soluble forms of NDB2 oxidised only NADH like the membrane-bound enzyme. In solution, the active StNDB1 resided as oligomers of dimeric units, mainly hexamers, and recombinant AtNDB2 was highly oligomeric. Within a physiological pH range, an acidic pH was found to lower the Ca2+ demand for activation of the mitochondrial and E. coli-produced NADPH oxidation via NDB1, as compared to a more alkaline pH. Depending on pH, 3-82 µM Ca2+ was needed. In contrast, the sub-µM Ca2+ demand for activation of NADH oxidation was not linked to pH. Both soluble and mitochondrial StNDB1 (NADPH oxidation) could respond quickly to increased and decreased Ca2+, whereas mitochondrial NADH oxidation responded quickly to Ca2+ increase but slowly to Ca2+ decrease. Overall, the results suggest that in vivo, the activity of NDB1 is rapidly controlled by pH-shift-associated Ca2+ spikes in the cytosol whereas NDB2 may be more continuously active. Based on modelling of NDB1, the core catalytic parts and dimerization surface showed distinct similarities to the structures of yeast ScNDI1 and Plasmodium falciparum PfNDH-2. This analysis highlighted motifs that correlate with NAD(P)H substrate specificity, and which were followed by evolutionary analysis. Most eukaryotic species have NDB proteins that contain a non-acidic motif for NADPH binding. Angiosperms and liverworts contain NDB proteins of NDB1- and NDB2- type, i.e. they contain acidic and non-acidic motifs for NADH and NADPH binding, respectively. This indicates that plants have more flexibility for external NAD(P)H oxidation as compared to other eukaryotes. Based on the evolutionary analysis, Ca2+-dependent external NADPH oxidation appears to be an ancient process as compared to NADH oxidation, and thus possibly has a more fundamental function in cellular redox metabolism.
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| 5. |
- Hao, Mengshu, et al.
(författare)
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The evolution of substrate specificity-associated residues and Ca2+-binding motifs in EF-hand-containing type II NAD(P)H dehydrogenases
- 2016
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Ingår i: Physiologia Plantarum. - : WILEY. - 0031-9317 .- 1399-3054. ; 157:3, s. 338-351
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Tidskriftsartikel (refereegranskat)abstract
- Most eukaryotic organisms, except some animal clades, have mitochondrial alternative electron transport enzymes that allow respiration to bypass the energy coupling in oxidative phosphorylation. The energy bypass enzymes in plants include the external type II NAD(P)H dehydrogenases (DHs) of the NDB family, which are characterized by an EF-hand domain for Ca2+ binding. Here we investigate these plant enzymes by combining molecular modeling with evolutionary analysis. Molecular modeling of the Arabidopsis thalianaAtNDB1 with the yeast ScNDI1 as template revealed distinct similarities in the core catalytic parts, and highlighted the interaction between the pyridine nucleotide and residues correlating with NAD(P)H substrate specificity. The EF-hand domain of AtNDB1 has no counterpart in ScNDI1, and was instead modeled with Ca2+-binding signal transducer proteins. Combined models displayed a proximity of the AtNDB1 EF-hand domain to the substrate entrance side of the catalytic part. Evolutionary analysis of the eukaryotic NDB-type proteins revealed ancient and recent reversions between the motif observed in proteins specific for NADH (acidic type) and NADPH (non-acidic type), and that the clade of enzymes with acidic motifs in angiosperms derives from non-acidic-motif NDB-type proteins present in basal plants, fungi and protists. The results suggest that Ca2+-dependent external NADPH oxidation is an ancient process, indicating that it has a fundamental importance for eukaryotic cellular redox metabolism. In contrast, the external NADH DHs in plants are products of a recent expansion, mirroring the expansion of the alternative oxidase family.
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| 6. |
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| 7. |
- Lu, Zijia, et al.
(författare)
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A polysaccharide utilization locus from Chitinophaga pinensis simultaneously targets chitin and β-glucans found in fungal cell walls
- 2023
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Ingår i: mSphere. - : American Society for Microbiology. - 2379-5042.
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Tidskriftsartikel (refereegranskat)abstract
- In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL’s outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade.
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| 8. |
- Lu, Zijia, et al.
(författare)
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Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis
- 2023
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 290:11, s. 2909-2922
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Tidskriftsartikel (refereegranskat)abstract
- The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.
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| 9. |
- Rasmusson, Allan G., et al.
(författare)
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Integrity Assessment of Isolated Plant Mitochondria
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; 2363, s. 51-62
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Bokkapitel (refereegranskat)abstract
- The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD+, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of mitochondria analysed. Here, we review the advantages and disadvantages of different integrity assays and present protocols for integrity assays that require relatively small amounts of mitochondria. They are based on the permeability of the outer membrane to cytochrome c, and the inner membrane to protons or NAD(H). The latter has the advantage of utilizing a membrane-bound activity (complex I) and the pore-forming peptide alamethicin to gain access to the matrix space. These methods together provide a toolbox for the determination of functionality and quality of isolated mitochondria.
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| 10. |
- Rasmusson, Allan G., et al.
(författare)
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Mitochondrial NAD(P)H oxidation pathways and nitrate/ammonium redox balancing in plants
- 2020
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Ingår i: Mitochondrion (Amsterdam. Print). - : ELSEVIER SCI LTD. - 1567-7249 .- 1872-8278. ; 53, s. 158-165
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Forskningsöversikt (refereegranskat)abstract
- Plant mitochondrial oxidative phosphorylation is characterised by alternative electron transport pathways with different energetic efficiencies, allowing turnover of cellular redox compounds like NAD(P)H. These electron transport chain pathways are profoundly affected by soil nitrogen availability, most commonly as oxidized nitrate (NO3-) and/or reduced ammonium (NH4+). The bioenergetic strategies involved in assimilating different N sources can alter redox homeostasis and antioxidant systems in different cellular compartments, including the mitochondria and the cell wall. Conversely, changes in mitochondrial redox systems can affect plant responses to N. This review explores the integration between N assimilation, mitochondrial redox metabolism, and apoplast metabolism.
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