| 1. |
- Engqvist, Martin, 1983, et al.
(författare)
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GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis
- 2015
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 169:2, s. 1042-1061
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Tidskriftsartikel (refereegranskat)abstract
- In roots of Arabidopsis (Arabidopsis thaliana), L-lactate is generated by the reduction of pyruvate via L-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative L-lactate-metabolizing enzymes based on their homology to CYB2, the L-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses L-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than L-lactate. The key factor making GOX3 more efficient with L-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize L-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-offunction and overexpressor plants indicate that L-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on L-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes L-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of L-lactate after its formation under normoxia.
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| 2. |
- Engqvist, Martin, 1983, et al.
(författare)
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Metabolic engineering of photorespiration
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; 1653, s. 137-155
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of two alternative glycolate catabolic pathways in the chloroplasts of Arabidopsis thaliana rendered plants with increased biomass. To introduce these synthetic pathways, the selected genes were stepwise integrated in the nuclear genome of wild-type plants. These plants were transformed by Agrobacterium tumefaciens carrying the binary vectors using the floral dip method. Selection of transformants was conducted using different selection agents and the expression of the transgenes was confirmed by PCR and enzyme activity measurements.
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| 3. |
- Engqvist, Martin, 1983, et al.
(författare)
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Mitochondrial 2-hydroxyglutarate metabolism
- 2014
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Ingår i: Mitochondrion. - : Elsevier BV. - 1567-7249. ; 19:Part B, s. 275-281
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Tidskriftsartikel (refereegranskat)abstract
- 2-Hydroxyglutarate (2-HG) is a five-carbon dicarboxylic acid with a hydroxyl group at the alpha position, which forms a stereocenter in this molecule. Although the existence of mitochondrial D- and L-2HG metabolisms has long been known in different eukaryotes, the biosynthetic pathways, especially in plants, have not been completely elucidated. While D-2HG is involved in intermediary metabolism, L-2HG may not have a cellular function but it needs to be recycled through a metabolic repair reaction. Independent of their metabolic origin, D- and L-2HG are oxidized in plant mitochondria to 2-ketoglutarate through the action of two stereospecific enzymes, d- and l-2-hydroxyacid dehydrogenases. While plants are to a large extent unaffected by high cellular concentrations of D-2HG, deficiencies in the metabolism of D- and L-2HG result in fatal disorders in humans. We present current data gathered on plant D- and L-2HG metabolisms and relate it to existing knowledge on 2HG metabolism in other organisms. We focus on the metabolic origin of these compounds, the mitochondrial catabolic steps catalyzed by the stereospecific dehydrogenases, and phylogenetic relationships between different studied 2-hydroxyacid dehydrogenases.
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| 4. |
- Engqvist, Martin, 1983, et al.
(författare)
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Plant D-2-hydroxyglutarate dehydrogenase participates in the catabolism of lysine especially during senescence
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:13, s. 11382-11390
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Tidskriftsartikel (refereegranskat)abstract
- D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH(d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism.
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| 5. |
- Engqvist, Martin, 1983, et al.
(författare)
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Two D-2-hydroxy-acid dehydrogenases in arabidopsis thaliana with catalytic capacities to participate in the last reactions of the methylglyoxal and β-oxidation pathways
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:37, s. 25026-25037
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Tidskriftsartikel (refereegranskat)abstract
- The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae D-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of D- and L-lactate, D-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for D-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for L-lactate and glycolate, respectively, and a Km value for D-lactate of ∼160 μM. Knock-out mutants showed impaired growth in the presence of D-lactate or methylglyoxal. Collectively, the data indicated that the protein is a D-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific D-2-hydroxyglutarate (D-2HG) conversion in the presence of an organic cofactor with a Km value of ∼580 μM. Thus, the enzyme was characterized as a D-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total D-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of D-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.
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| 6. |
- Hüdig, M., et al.
(författare)
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Biochemical control systems for small molecule damage in plants
- 2018
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Ingår i: Plant Signaling and Behavior. - : Informa UK Limited. - 1559-2316 .- 1559-2324. ; 13:5
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Forskningsöversikt (refereegranskat)abstract
- As a system, plant metabolism is far from perfect: small molecules (metabolites, cofactors, coenzymes, and inorganic molecules) are frequently damaged by unwanted enzymatic or spontaneous reactions. Here, we discuss the emerging principles in small molecule damage biology. We propose that plants evolved at least three distinct systems to control small molecule damage: (i) repair, which returns a damaged molecule to its original state; (ii) scavenging, which converts reactive molecules to harmless products; and (iii) steering, in which the possible formation of a damaged molecule is suppressed. We illustrate the concept of small molecule damage control in plants by describing specific examples for each of these three categories. We highlight interesting insights that we expect future research will provide on those systems, and we discuss promising strategies to discover new small molecule damage-control systems in plants.
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| 7. |
- Hüdig, M., et al.
(författare)
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Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehydrogenase
- 2014
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 56:9, s. 1820-1830
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild. © 2015 The Author 2015.
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| 8. |
- Kuhn, A., et al.
(författare)
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D-2-hydroxyglutarate metabolism is linked to photorespiration in the shm1-1 mutant
- 2013
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Ingår i: Plant Biology. - : Wiley. - 1435-8603. ; 15:4, s. 776-784
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Tidskriftsartikel (refereegranskat)abstract
- The Arabidopsis mutant shm1-1 is defective in mitochondrial serine hydroxymethyltransferase 1 activity and displays a lethal photorespiratory phenotype at ambient CO2 concentration but grows normally at high CO2. After transferring high CO2-grown shm1-1 plants to ambient CO2, the younger leaves remain photosynthetically active while developed leaves display increased yellowing and decreased FV/FM values. Metabolite analysis of plants transferred from high CO2 to ambient air indicates a massive light-dependent (photorespiratory) accumulation of glycine, 2-oxoglutarate (2OG) and D-2-hydroxyglutarate (D-2HG). Amino acid markers of senescence accumulated in ambient air in wild-type and shm1-1 plants maintained in darkness and also build up in shm1-1 in the light. This, together with an enhanced transcription of the senescence marker SAG12 in shm1-1, suggests the initiation of senescence in shm1-1 under photorespiratory conditions. Mitochondrial D-2HG dehydrogenase (D-2HGDH) converts D-2HG into 2OG. In vitro studies indicate that 2OG exerts competitive inhibition on D-2HGDH with a Ki of 1.96 mm. 2OG is therefore a suitable candidate as inhibitor of the in vivo D-2HGDH activity, as 2OG is produced and accumulates in mitochondria. Inhibition of the D-2HGDH by 2OG is likely a mechanism by which D-2HG accumulates in shm1-1, however it cannot be ruled out that D-2HG may also accumulate due to an active senescence programme that is initiated in these plants after transfer to photorespiratory conditions. Thus, a novel interaction of the photorespiratory pathway with cellular processes involving D-2HG has been identified.
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| 9. |
- Maier, A., et al.
(författare)
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Transgenic introduction of a glycolate oxidative cycle into A. thaliana chloroplasts leads to growth improvement
- 2012
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 3:FEB
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Tidskriftsartikel (refereegranskat)abstract
- The photorespiratory pathway helps illuminated C3-plants under conditions of limited CO2availability by effectively exporting reducing equivalents in form of glycolate out of the chloroplast and regenerating glycerate-3-P as substrate for RubisCO. On the other hand, this pathway is considered as probably futile because previously assimilated CO2 is released in mitochondria. Consequently, a lot of effort has been made to reduce this CO2 loss either by reducing fluxes via engineering RubisCO or circumventing mitochondrial CO2 release by the introduction of new enzyme activities. Here we present an approach following the latter route, introducing a complete glycolate catabolic cycle in chloroplasts ofArabidopsis thalianacomprising glycolate oxidase (GO), malate synthase (MS), and catalase (CAT). Results from plants bearing both GO and MS activities have already been reported. This previous work showed that the H2O2produced by GO had strongly negative effects. These effects can be prevented by introducing a plastidial catalase activity, as reported here. Transgenic lines bearing all three transgenic enzyme activities were identified and some with higher CAT activity showed higher dry weight, higher photosynthetic rates, and changes in glycine/serine ratio compared to the wild type. This indicates that the fine-tuning of transgenic enzyme activities in the chloroplasts seems crucial and strongly suggests that the approach is valid and that it is possible to improve the growth of A. thaliana by introducing a synthetic glycolate oxidative cycle into chloroplasts.
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| 10. |
- Peterhansel, C., et al.
(författare)
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Engineering photorespiration : current state and future possibilities
- 2013
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Ingår i: Plant Biology. - : Wiley-Blackwell. - 1435-8603 .- 1438-8677. ; 15:4, s. 754-758
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Forskningsöversikt (refereegranskat)abstract
- Reduction of flux through photorespiration has been viewed as a major way to improve crop carbon fixation and yield since the energy-consuming reactions associated with this pathway were discovered. This view has been supported by the biomasses increases observed in model species that expressed artificial bypass reactions to photorespiration. Here, we present an overview about the major current attempts to reduce photorespiratory losses in crop species and provide suggestions for future research priorities.
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| 11. |
- Pires, Marcel V., et al.
(författare)
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The influence of alternative pathways of respiration that utilize branched-chain amino acids following water shortage in Arabidopsis
- 2016
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Ingår i: Plant, Cell and Environment. - : Wiley. - 1365-3040 .- 0140-7791. ; 39:6, s. 1304-1319
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Tidskriftsartikel (refereegranskat)abstract
- During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response.
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| 12. |
- Wienstroer, J., et al.
(författare)
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D-Lactate dehydrogenase as a marker gene allows positive selection of transgenic plants
- 2012
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 586:1, s. 36-40
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Tidskriftsartikel (refereegranskat)abstract
- d-Lactate negatively affects Arabidopsis thaliana seedling development in a concentration-dependent manner. At media d-lactate concentrations greater than 5-10 mM the development of wild-type plants is arrested shortly after germination whereas plants overexpressing the endogenous d-lactate dehydrogenase (d-LDH) detoxify d-lactate to pyruvate and survive. When the transgenic plants are further transferred to normal growth conditions they develop indistinguishably from the wild type. Thus, d-LDH was successfully established as a new marker in A. thaliana allowing selecting transgenic plants shortly after germination. The selection on d-lactate containing media adds a new optional marker system, which is especially useful if the simultaneous selection of multiple constructs is desired.
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