Emerging concepts in the flavinylation of succinate dehydrogenase

Kim, Hyung J.; Winge, Dennis R.

doi:10.1016/j.bbabio.2013.01.012

Cited by 60 publications

(51 citation statements)

References 67 publications

(149 reference statements)

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“…17 From these combined results, it has been proposed that SdhE/5 interacts with SdhA/1 to mediate flavinylation, yet the molecular details of this process remain unresolved. 15,18 To examine the molecular mechanism of SdhE-dependent flavinylation of SdhA, this study describes truncation and sitedirected mutagenesis of residues conserved across bacterial homologues of SdhE. Using phenotype rescue assays, we demonstrated that a highly conserved RGxxE motif is required for flavinylation and activation of SDH activity.…”

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confidence: 98%

The Conserved RGxxE Motif of the Bacterial FAD Assembly Factor SdhE Is Required for Succinate Dehydrogenase Flavinylation and Activity

McNeil

Fineran

2013

Biochemistry

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Succinate dehydrogenase (SDH) is an important respiratory enzyme that plays a critical role in the generation of energy in the majority of eukaryotes, bacteria, and archaea. The activity of SDH is dependent on the covalent attachment of the redox cofactor FAD to the flavoprotein subunit SdhA. In the Gram-negative bacteria Escherichia coli and Serratia sp. ATCC 39006, the covalent attachment of FAD to SdhA is dependent on the FAD assembly factor SdhE (YgfY). Although mechanisms have been proposed, experimental evidence that elucidates the molecular details of SdhE-mediated flavinylation are scarce. In this study, truncation and alanine swap mutagenesis of SdhE identified a highly conserved RGxxE motif that was important for SdhE function. Interestingly, RGxxE site-directed variants were not impaired in terms of protein folding or interactions with SdhA. Purification and analysis of SdhA from different mutant backgrounds demonstrated that SdhE interacts with and flavinylates folded SdhA without a requirement for the assembly of the entire SDH complex. SdhA was also partially active in the absence of SdhE, suggesting that SdhA is able to attach FAD through an inefficient autocatalytic mechanism. The results presented are of widespread relevance because SdhE and SDH are required for bacterial pathogenesis and mutations in the eukaryotic homologues of SdhE and SDH are associated with cancer in humans.

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confidence: 98%

The Conserved RGxxE Motif of the Bacterial FAD Assembly Factor SdhE Is Required for Succinate Dehydrogenase Flavinylation and Activity

McNeil

Fineran

2013

Biochemistry

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“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 [17], (c) [68], (d) [69], (e) [70], (f) [71], (g) [72], (h) [73], (i) [74], (j) [75], (k) [76], (l) [77], (m) [22], (n) [29], (o) [78], (p) [58], (q) [79], (r)[34] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17 ((a) [48], (b) [45], (c) [47], (d) [43], (e) [44], (f) [39], (g) …”

Section: Resultsunclassified

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review

et al. 2014

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Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratorydeficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes. Opposed Reviewers:Claire Remacle -Génétique des microorganismes -Institut de Botanique B22 Boulevard du Rectorat, 27 -Université de Liège-B-4000 Liège, Belgique Tel +32 4 3663812 -Fax +32 4 3663840 e-mail: c.remacle@ulg.ac.be First, we wish to thank the two anonymous reviewers for their constructive comments. We found their suggestions very helpful to improve the quality of our manuscript. We have addressed all the points they raised and have modified the text accordingly. A detailed response to reviewers' comments is included below.We believe we have addressed all referees' comments and we hope this revised version is now suitable for publication in Biochimie.We are looking forward to hearing from you, With best regards, Cover LetterResponse to reviewers The text has been modified in order to give more background details on the methodology used to create and screen respiratory mutants in Chlamydomonas. The main modifications correspond to: Paragraph 2.3: -p8. A figure (Fig. 4) has been added in order to provide more information on dark-dier/slow growth screens for different classes of mutant.-p9. The mitochondrial transformation method used in Chlamydomonas has been detailed.Paragraph 2.4: -p10. The TTC-based screen has been explained.Finally, all along the text the methods used to create nuclear and mitochondrial mutants has been indicated. Also, the text could benefit from being proof-read by a native English speaker as the grammar is a little awkward in places. I highlight a couple of points below, but there are a number of other places where the English could be improved for clarity.The text has been proof-read by a good English speaker as requested and the text has been modified according to the comments.Minor points:1. Abstract, line 1: "the genetics" have been replaced by "genetic manipu...

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“…NADH is localized mainly in the mitochondria and participates predominantly in cellular energy metabolism, while FAD is contained in both cytoplasm and mitochondria and is involved, apart from oxidative phosphorylation, in various biochemical processes (glutathione utilization, lipogenesis, lipid peroxidation, antioxidant reactions, acetyl coenzyme A synthesis, the glutathione-ascorbate cycle, the pentose phosphate cycle), which considerably complicates data analysis [4,13,21,27].…”

Section: Cofactors Nadn and Fad And Their Role In Cellular Energy Metmentioning

confidence: 99%

Metabolic Imaging in the Study of Oncological Processes (Review)

Lukina¹,

Shirmanova²,

Sergeeva³

et al. 2016

Sovrem Tehnol Med

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There have been reviewed the main approaches to the study of energy metabolism in cancer cells, based on fluorescent imaging of NADH and FAD cofactors. The term "metabolic imaging" covers a range of modern fluorescent techniques for detecting NADH and FAD according to fluorescence intensity and/or lifetime. These cofactors play an important role in energy metabolism reactions acting as electron carriers and, being fluorescent, serve as a basis for metabolic process analysis in living cells and tissues without the use of additional coloring agents. Particular attention is paid to metabolic changes associated with carcinogenesis. Numerous examples of metabolic imaging application in cell cultures in vitro, animal and human tumors in vivo, as well as in patients' tumor biopsy samples have demonstrated its being highly demanded for biomedical research in the area of oncology.Key words: metabolic imaging; energy metabolism; fluorescence lifetime; redox ratio; NADH; FAD; oxidative phosphorylation; glycolysis; tumor cells.For contacts: Mariya M. lukina, e-mail: kuznetsova.m.m@yandex.ru Cells need energy to maintain homeostasis. All processes of cell activity, such as generating concentration gradients, cytoskeleton movement, DNA repair, transcription, translation and vesicular transport, are energy-dependent. Energy metabolism of normal cells is known to differ significantly from cell metabolism in case of a disease. Therefore, the metabolic status can serve as an indicator for diagnosis and visualization of response to pathological process treatment. Taking into account high incidence of oncological diseases, assessment of metabolic phenotype of tumor cells is particularly urgent.Development of optical visualization technologies has provided the possibility of noninvasive analysis of metabolic cofactors NADH and FAD in living tumor cells at high spatial resolution (up to several hundred Metabolic Imaging in the Study of Oncological Process nanometers) without using additional coloring agents and with no significant effect on biochemical and physiological condition of cells. The term "metabolic imaging" covers a range of modern fluorescent techniques which allow visualizing NADH and FAD according to their fluorescence intensity and/or lifetime.The present review characterizes the properties of energy metabolism in cancer cells, describes the two key approaches to the assessment of metabolic status: analysis of cofactor fluorescence intensity ratio (redox ratio) and their fluorescence lifetime. There have been presented numerous examples of metabolic imaging application in cell cultures in vitro, animal and human tumors in vivo, as well as in patients' tumor biopsy samples.

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Emerging concepts in the flavinylation of succinate dehydrogenase

Cited by 60 publications

References 67 publications

The Conserved RGxxE Motif of the Bacterial FAD Assembly Factor SdhE Is Required for Succinate Dehydrogenase Flavinylation and Activity

The Conserved RGxxE Motif of the Bacterial FAD Assembly Factor SdhE Is Required for Succinate Dehydrogenase Flavinylation and Activity

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review

Metabolic Imaging in the Study of Oncological Processes (Review)

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