Expression and Characterization of Two Pathogenic Mutations in Human Electron Transfer Flavoprotein

Salazar, Denise; Zhang, Lening; Degala, Gregory D.; Frerman, Frank E.

doi:10.1074/jbc.272.42.26425

Cited by 43 publications

(42 citation statements)

References 34 publications

(46 reference statements)

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“…The total buried interfacial surface visible for the crystallographically ordered atoms is 536 Å 2 and covers only 4.3 and 3.2% of the accessible surface of an MCAD monomer and ETF, respectively. That this interaction surface is rather small, albeit highly complementary (shape correlation statistic (15) of 0.704), is consistent with kinetic data that suggest a weakly interacting system (16,17).…”

Section: Resultssupporting

confidence: 63%

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

Toogood

Thiel

Basran

et al. 2004

Journal of Biological Chemistry

137

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The crystal structure of the human electron transferring flavoprotein (ETF)⅐medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF⅐MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD⅐ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu 212 and ETF Arg␣ 249 are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

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Section: Resultssupporting

confidence: 63%

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

Toogood

Thiel

Basran

et al. 2004

Journal of Biological Chemistry

137

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“…This is likely to promote the re-oxidation of the anionic hydroquinone to fully oxidized FAD. The Thr 169 O ␥1 -FAD N5/O4 hydrogen bonds described here are also observed in human electron transfer flavoprotein (ETF) (29), where the enzyme variant T266M is pathogenic and appears to specifically affect the oxidative half-reaction between ETF and ETF-QO (30).…”

Section: Table 3 Statistics For Data Collection and Crystallographic mentioning

confidence: 72%

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Kujawa

Ebner

Leitner

et al. 2006

Journal of Biological Chemistry

235

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Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H 2 O 2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose-and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k cat , and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Pyranose 2-oxidase (P2Ox, 3 pyranose:oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) is a flavin adenine dinucleotide (FAD)-dependent oxidase present in the hyphal periplasmic space (1) of wood-degrading basidiomycetes (2, 3). These fungi are the only known microorganisms that are capable of fully mineralizing lignin, and P2Ox has a proposed role in the oxidative events (4) of lignin degradation by providing the essential co-substrate, H 2 O 2 , for lignin and manganese peroxidases (5, 6). An alternative hypothesis assigns a role for P2Ox in both H 2 O 2 production and in the reduction of quinones in the periplasm or in the extracellular environment (7). P2Ox from the white-rot fungi Trametes multicolor (Trametes ochracea) and Peniophora gigantea are hitherto the most studied biochemically (7-10) and structurally (11, 12).P2Ox oxidizes a broad range of carbohydrate substrates that are natural constituents of hemicelluloses, allowing most lignocellulose-derived sugars to be utilized. Substrates can be oxidized regioselectively at the C2 position, although some oxidation at C3 can occur as a side reaction (10). For C2 oxidation, D-glucose, D-xylose, and L-sorbose are good or reasonably good substrates, and D-galactose and L-arabinose perform poorly as substrates (7). Based on the catalytic efficiency, k cat /K m , D-glucose (D-Glc) is the best substrate for T. multicolor P2Ox (7). Substrates that are oxidized at C3 were analyzed for P. gigantea P2Ox and include 2-deoxy-D-glucose, 2-keto-D-glucose, and methyl ␤-D-glucosides (13, 10). That oxidation can take place either at C2 or at C3 presupposes two distinct, productive binding modes (referred to here as C2 ox and C3 ox ) for a monosaccharide in the P2Ox active site.P2Ox from T. multicolor is homotetrameric with a molecular mass of 270 kDa (7) where each of the four subunits carries one FAD molecule bound covalently to N ⑀2 (i.e. N3) of His 167 via its 8␣-methyl group (14, 11). The...

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“…Defects in ETF are responsible for causing the inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired (54). Although not directly involved in binding the flavin adenine dinucleotide cofactor of ETF (51), introduction of 3-NT could destabilize the tertiary structure of domain I of this protein.…”

Section: Discussionmentioning

confidence: 99%

Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging

Kański¹,

Behring²,

Pelling³

et al. 2005

American Journal of Physiology-Heart and Circulatory Physiology

207

160

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Proteomic techniques were used to identify cardiac proteins from whole heart homogenate and heart mitochondria of Fisher 344/Brown Norway F1 rats, which suffer protein nitration as a consequence of biological aging. Soluble proteins from young (5 mo old) and old (26 mo old) animals were separated by one-and two-dimensional gel electrophoresis. One-and two-dimensional Western blots with an anti-nitrotyrosine antibody show an age-related increase in the immunoresponse of a few specific proteins, which were identified by nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS). Complementary proteins were immunoprecipitated with an immobilized anti-nitrotyrosine antibody followed by NSI-MS/MS analysis. A total of 48 proteins were putatively identified. Among the identified proteins were ␣-enolase, ␣-aldolase, desmin, aconitate hydratase, methylmalonate semialdehyde dehydrogenase, 3-ketoacyl-CoA thiolase, acetyl-CoA acetyltransferase, GAPDH, malate dehydrogenase, creatine kinase, electron-transfer flavoprotein, manganese-superoxide dismutase, F1-ATPase, and the voltage-dependent anion channel. Some contaminating blood proteins including transferrin and fibrinogen ␤-chain precursor showed increased levels of nitration as well. MS/MS analysis located nitration at Y105 of the electron-transfer flavoprotein. Among the identified proteins, there are important enzymes responsible for energy production and metabolism as well as proteins involved in the structural integrity of the cells. Our results are consistent with agedependent increased oxidative stress and with free radical-dependent damage of proteins. Possibly the oxidative modifications of the identified proteins contribute to the age-dependent degeneration and functional decline of heart proteins.heart; mitochondria THERE IS INCREASING EVIDENCE for an age-dependent decline of cardiac performance (45). Several studies show this performance decline to be associated with oxidative stress (42, 43, 57, 58), i.e., elevated levels of reactive oxygen species. For example, various biomarkers of oxidative stress such as oxo-2-deoxyguanosine, H 2 O 2 , 3-nitrotyrosine (3-NT), N-⑀-methyllysine, malondialaldehyde, and advanced glycation end products (14,22,47,48,55,62) increase with the age in cardiac tissue. Moreover, dietary restriction significantly reduces the age-dependent accumulation of these oxidative markers in heart, which indicates the importance of oxidative stress in cardiac aging (46).A causal role of reactive oxygen species in the age-dependent decline of cardiac dysfunction can, however, only be defined if biological targets of these species are identified and the physiological impact of biomolecular modification is characterized. A first step to establish molecular mechanisms of age-dependent cardiac dysfunction is the proteomic identification of oxidatively modified proteins. Various studies have indicated the formation of reactive nitrogen species in acute myocardiac disorders such as heart failure (1, 28, 31). These reactive nitrogen species are met...

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Expression and Characterization of Two Pathogenic Mutations in Human Electron Transfer Flavoprotein

Cited by 43 publications

References 34 publications

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging

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