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Characterization of metabolic steps involved in the biosynthesis of the precursor of coenzyme Q in Saccharomyces cerevisiae
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Abstracts
thereby, regulating the communication between mitochondria and cytosol. This protein is considered to be a key player in mitochondriameditated apoptosis via involvement in opening of mitochondrial permeability transition pore (PTP) and interactions with pro-apoptotic outer membrane proteins such as hexokinases and Bcl-2 family proteins. Although it is still a matter of debate whether VDAC is directly involved in PTP opening, there have been several studies indicating that VDAC interacts with various low-molecular weight chemicals, some of which were proven to regulate the PTP opening and/or apoptotic pathways. Ubiquinone-0 (UQ0), a member of short-chain UQ, is one of the most potent inhibitor of PTP opening. However, despite the numerous attempts to identify its molecular target, there is no experimental data showing the direct interaction between UQ and a specific mitochondrial protein. In order to clarify the interaction between UQ and mitochondrial protein(s), we performed photoaffinity labeling experiments using photoreactive UQ probes with the isolated mitochondria from Saccharomyces cerevisiae. Based on careful biochemical (in combination with click chemistry) and proteomic analyses, we revealed that UQ probes specifically bind to mitochondrial VDAC. The labeling was markedly suppressed in the presence of an excess amount of short-chain UQs such as UQ0 and UQ2. The present study, for the first time, demonstrates that mitochondrial VDAC specifically accommodates a quinone-head ring of UQ. doi:10.1016/j.bbabio.2016.04.144
04.10 Characterization of metabolic steps involved in the biosynthesis of the precursor of coenzyme Q in Saccharomyces cerevisiae Laurie-Anne Payeta, Mélanie Lerouxa, John C. Willisonb, Akio Kiharac, Ludovic Pelosia, Fabien Pierrela, a TIMC-IMAG laboratory, Univ. Grenoble Alpes-CNRS, Grenoble France b CEA-Grenoble, DRF-BIG-CBM, Grenoble France c Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo Japan E-mail address: [email protected] (F. Pierrel) The redox lipid coenzyme Q (Q) is essential for oxidative phosphorylation in mitochondria which provide most of the energy to heterotrophic eukaryotic cells. As such, mutations in several genes required for the biosynthesis of Q cause heterogeneous disorders known as primary Q deficiency [1]. Despite the discovery of Q almost sixty years ago [2], several aspects of its biosynthesis are still unknown, in particular the pathway that produces 4-hydroxybenzoic acid (4-HB), the aromatic precursor of Q [3]. By combining genetic and biochemical approaches in the yeast Saccharomyces cerevisiae, we have uncovered the first and last steps of the pathway: the deamination of tyrosine to 4-hydroxyphenylpyruvate by the Aro8 and Aro9 proteins, and the oxidation of 4-hydroxybenzaldehyde to 4-HB by the aldehyde dehydrogenase Hfd1. Inactivation of the hfd1 gene resulted in Q deficiency and the related human enzyme ALDH3A1 was found to catalyze the oxidation of 4-hydroxybenzaldehyde in yeast. Our results also demonstrate that S. cerevisiae does not possess a chorismatase to synthesize 4-HB directly from chorismic acid, which is the pathway used in bacteria. Overall, our study represents the first characterization of the eukaryotic metabolic pathway that produces the precursor of Q from tyrosine and points to gene candidates that may participate to the biosynthesis of 4-HB in humans. References 1. M.A. Desbats, G. Lunardi, M. Doimo, E. Trevisson, L. Salviati, Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency, J Inherit Metab Dis, 38 (2015) 145–156.
2. F.L. Crane, Y. Hatefi, R.L. Lester, C. Widmer, Isolation of a quinone from beef heart mitochondria, Biochim. Biophys. Acta, 25 (1957) 220–221. 3. M. Kawamukai, Biosynthesis of coenzyme Q in eukaryotes, Bioscience Biotechnology and Biochemistry, 80 (2016) 23–33. doi:10.1016/j.bbabio.2016.04.145
04.11 A single quinol oxidation site in E. coli nitrate reductase A reacts with the three endogenous respiratory quinones Julia Rendon, Eric Pilet, Maryam Seif-Eddine, Bruno Guigliarelli, Axel Magalon, Stephane Grimaldi Unité de Bioénergétique et Ingénierie des Protéines, Aix Marseille Université & CNRS, Marseille France Laboratoire de Chimie Bactérienne, Aix Marseille Université & CNRS, Marseille France Corresponding author at: Unité de Bioénergétique et Ingénierie des Protéines, Aix Marseille Université & CNRS, Marseille France. E-mail address: [email protected] (S. Grimaldi) Among prokaryotes, γ-proteobacteria are unique due to their ability to synthesize quinones of low, intermediate and high-redox midpoint potentials. This property allows them to more easily switch between low- and high-potential electron transport chains. A best known example is the gut bacterium Escherichia coli able to use oxygen, nitrate, nitrite, fumarate, S- or N-oxides or even selenate as terminal electron acceptors. A key question is how these electron transport chains make use of different quinone species. To decipher the influence of the protein environment in tuning enzyme reactivity towards quinones, we use E. coli nitrate reductase A (NarGHI), a membrane-bound quinol oxidizing enzyme, as a model system. As such, we have previously shown that NarGHI can stabilize menasemiquinones or ubisemiquinones within its quinol oxidation site QD. We provide here experimental evidences for the efficient use by NarGHI of demethylmenaquinone which predominates under nitrate respiring conditions [1]. We report the stabilization of demethylmenasemiquinone within the NarGHI QD site. In addition, we have elucidated the origin of the partially resolved structure specifically detected on its EPR spectrum, as well as its peculiar binding mode to the protein which involves a single short hydrogen bond to a histidine residue. Overall, the nitrate reductase complex constitutes an ideal system to address at the molecular level how a single protein site tunes the reactivity towards three different endogenous quinones. References 1. J. Rendon, E. Pilet, Z. Fahs, F. Seduk, L. Sylvi, M.H. Chehade, F. Pierrel, B. Guigliarelli, A. Magalon, S. Grimaldi, Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site, Biochimica et Biophysica Acta-Bioenergetics, 1847 (2015) 739–747. doi:10.1016/j.bbabio.2016.04.146
04.12 The idebenone metabolite QS10 is an electron donor to complex III and rescues respiration in complex I-deficient cells and rotenone-treated zebrafish Marco Schiavonea,, Valentina Giorgioa, Valeria Petronillia, Francesco Argentonb, Tatiana Da Rosc, Maurizio Pratoc, Paolo Bernardia a Department of Biomedical Sciences, University of Padova and Consiglio Nazionale delle Ricerche Neuroscience Institute, Padova Italy b Department of Biology, University of Padova, Padova Italy
Abstracts
thereby, regulating the communication between mitochondria and cytosol. This protein is considered to be a key player in mitochondriameditated apoptosis via involvement in opening of mitochondrial permeability transition pore (PTP) and interactions with pro-apoptotic outer membrane proteins such as hexokinases and Bcl-2 family proteins. Although it is still a matter of debate whether VDAC is directly involved in PTP opening, there have been several studies indicating that VDAC interacts with various low-molecular weight chemicals, some of which were proven to regulate the PTP opening and/or apoptotic pathways. Ubiquinone-0 (UQ0), a member of short-chain UQ, is one of the most potent inhibitor of PTP opening. However, despite the numerous attempts to identify its molecular target, there is no experimental data showing the direct interaction between UQ and a specific mitochondrial protein. In order to clarify the interaction between UQ and mitochondrial protein(s), we performed photoaffinity labeling experiments using photoreactive UQ probes with the isolated mitochondria from Saccharomyces cerevisiae. Based on careful biochemical (in combination with click chemistry) and proteomic analyses, we revealed that UQ probes specifically bind to mitochondrial VDAC. The labeling was markedly suppressed in the presence of an excess amount of short-chain UQs such as UQ0 and UQ2. The present study, for the first time, demonstrates that mitochondrial VDAC specifically accommodates a quinone-head ring of UQ. doi:10.1016/j.bbabio.2016.04.144
04.10 Characterization of metabolic steps involved in the biosynthesis of the precursor of coenzyme Q in Saccharomyces cerevisiae Laurie-Anne Payeta, Mélanie Lerouxa, John C. Willisonb, Akio Kiharac, Ludovic Pelosia, Fabien Pierrela, a TIMC-IMAG laboratory, Univ. Grenoble Alpes-CNRS, Grenoble France b CEA-Grenoble, DRF-BIG-CBM, Grenoble France c Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo Japan E-mail address: [email protected] (F. Pierrel) The redox lipid coenzyme Q (Q) is essential for oxidative phosphorylation in mitochondria which provide most of the energy to heterotrophic eukaryotic cells. As such, mutations in several genes required for the biosynthesis of Q cause heterogeneous disorders known as primary Q deficiency [1]. Despite the discovery of Q almost sixty years ago [2], several aspects of its biosynthesis are still unknown, in particular the pathway that produces 4-hydroxybenzoic acid (4-HB), the aromatic precursor of Q [3]. By combining genetic and biochemical approaches in the yeast Saccharomyces cerevisiae, we have uncovered the first and last steps of the pathway: the deamination of tyrosine to 4-hydroxyphenylpyruvate by the Aro8 and Aro9 proteins, and the oxidation of 4-hydroxybenzaldehyde to 4-HB by the aldehyde dehydrogenase Hfd1. Inactivation of the hfd1 gene resulted in Q deficiency and the related human enzyme ALDH3A1 was found to catalyze the oxidation of 4-hydroxybenzaldehyde in yeast. Our results also demonstrate that S. cerevisiae does not possess a chorismatase to synthesize 4-HB directly from chorismic acid, which is the pathway used in bacteria. Overall, our study represents the first characterization of the eukaryotic metabolic pathway that produces the precursor of Q from tyrosine and points to gene candidates that may participate to the biosynthesis of 4-HB in humans. References 1. M.A. Desbats, G. Lunardi, M. Doimo, E. Trevisson, L. Salviati, Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency, J Inherit Metab Dis, 38 (2015) 145–156.
2. F.L. Crane, Y. Hatefi, R.L. Lester, C. Widmer, Isolation of a quinone from beef heart mitochondria, Biochim. Biophys. Acta, 25 (1957) 220–221. 3. M. Kawamukai, Biosynthesis of coenzyme Q in eukaryotes, Bioscience Biotechnology and Biochemistry, 80 (2016) 23–33. doi:10.1016/j.bbabio.2016.04.145
04.11 A single quinol oxidation site in E. coli nitrate reductase A reacts with the three endogenous respiratory quinones Julia Rendon, Eric Pilet, Maryam Seif-Eddine, Bruno Guigliarelli, Axel Magalon, Stephane Grimaldi Unité de Bioénergétique et Ingénierie des Protéines, Aix Marseille Université & CNRS, Marseille France Laboratoire de Chimie Bactérienne, Aix Marseille Université & CNRS, Marseille France Corresponding author at: Unité de Bioénergétique et Ingénierie des Protéines, Aix Marseille Université & CNRS, Marseille France. E-mail address: [email protected] (S. Grimaldi) Among prokaryotes, γ-proteobacteria are unique due to their ability to synthesize quinones of low, intermediate and high-redox midpoint potentials. This property allows them to more easily switch between low- and high-potential electron transport chains. A best known example is the gut bacterium Escherichia coli able to use oxygen, nitrate, nitrite, fumarate, S- or N-oxides or even selenate as terminal electron acceptors. A key question is how these electron transport chains make use of different quinone species. To decipher the influence of the protein environment in tuning enzyme reactivity towards quinones, we use E. coli nitrate reductase A (NarGHI), a membrane-bound quinol oxidizing enzyme, as a model system. As such, we have previously shown that NarGHI can stabilize menasemiquinones or ubisemiquinones within its quinol oxidation site QD. We provide here experimental evidences for the efficient use by NarGHI of demethylmenaquinone which predominates under nitrate respiring conditions [1]. We report the stabilization of demethylmenasemiquinone within the NarGHI QD site. In addition, we have elucidated the origin of the partially resolved structure specifically detected on its EPR spectrum, as well as its peculiar binding mode to the protein which involves a single short hydrogen bond to a histidine residue. Overall, the nitrate reductase complex constitutes an ideal system to address at the molecular level how a single protein site tunes the reactivity towards three different endogenous quinones. References 1. J. Rendon, E. Pilet, Z. Fahs, F. Seduk, L. Sylvi, M.H. Chehade, F. Pierrel, B. Guigliarelli, A. Magalon, S. Grimaldi, Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site, Biochimica et Biophysica Acta-Bioenergetics, 1847 (2015) 739–747. doi:10.1016/j.bbabio.2016.04.146
04.12 The idebenone metabolite QS10 is an electron donor to complex III and rescues respiration in complex I-deficient cells and rotenone-treated zebrafish Marco Schiavonea,, Valentina Giorgioa, Valeria Petronillia, Francesco Argentonb, Tatiana Da Rosc, Maurizio Pratoc, Paolo Bernardia a Department of Biomedical Sciences, University of Padova and Consiglio Nazionale delle Ricerche Neuroscience Institute, Padova Italy b Department of Biology, University of Padova, Padova Italy