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Milagros Medina - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis fnrl protein is an NADPH dependent chloroplast oxidoreductase resembling bacterial ferredoxin nadp reductases
Physiologia Plantarum, 2018Co-Authors: Minna M Koskela, Kathe M Dahlstrom, Guillermina Goni, Nina Lehtimaki, Markus Nurmi, Adrian Velazquezcampoy, Guy T Hanke, Bettina Bolter, Tiina A Salminen, Milagros MedinaAbstract:Plastidic ferredoxin-NADP(+) oxidoreductases (FNRs;EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of flavin adenine dinucleotide (FAD) dependent oxidoreductases. In this study, we show that the Arabidopsis thaliana At1g15140 [Ferredoxin-NADP(+) oxidoreductase-like (FNRL)] is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with k(cat)=3.2 +/- 0.2s(-1), K-m(NADPH)=1.6 +/- 0.3M and k(cat)/K-m(NADPH)=2.0 +/- 0.4M(-1)s(-1). Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1, which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. In addition, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded -sheet and one -helix, and a C-terminal NADP(+)-binding / domain with a five-stranded -sheet with a pair of -helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs.
Takeshi Sakurai - One of the best experts on this subject based on the ideXlab platform.
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kinetics of nadp NADPH reduction oxidation catalyzed by the ferredoxin nad p reductase from the green sulfur bacterium chlorobaculum tepidum
Photosynthesis Research, 2016Co-Authors: Daisuke Seo, Takeshi Sakurai, Masaharu Kitashima, Kazuhito InoueAbstract:Ferredoxin-NAD(P)+ oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum (CtFNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. CtFNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of CtFNR with NADP+, NADPH, and (4S-2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNRox with NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460 nm within 1 ms, and then the absorbance further decreased gradually. The magnitude of the decrease increased with increasing NADPH concentration, but even with ~50-fold molar excess NADPH, the absorbance change was only ~45 % of that expected for fully reduced protein. The absorbance in the charge transfer (CT) band centered around 600 nm increased rapidly within 1 ms, then slowly decreased to about 70 % of the maximum. When CtFNRred was mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNRox with an apparent rate of ~4 s−1, whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between CtFNR and NADP+/NADPH is reversible, in accordance with its physiological function.
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pre steady state kinetic studies of redox reactions catalysed by bacillus subtilis ferredoxin nadp oxidoreductase with nadp NADPH and ferredoxin
Biochimica et Biophysica Acta, 2016Co-Authors: Takahiro Soeta, Hidehiro Sakurai, Pierre Setif, Takeshi SakuraiAbstract:Abstract Ferredoxin-NADP+ oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP+, NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP+ and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNRox)-NADPH and reduced FNR (FNRred)-NADP+, both having CT absorption bands centered at approximately 600 nm. After mixing BsFNRox with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNRred was mixed with NADP+, the amount of BsFNRox increased with increasing NADP+ concentration, but BsFNRred remained as the major species at equilibrium even with about 50-fold molar excess NADP+. In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~ 500 s− 1) was much higher than the reverse one (
Kathy K Griendling - One of the best experts on this subject based on the ideXlab platform.
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role of nadh NADPH oxidase derived h2o2 in angiotensin ii induced vascular hypertrophy
Hypertension, 1998Co-Authors: A M Zafari, Masuko Ushiofukai, M Akers, Ankoor S Shah, David G Harrison, W R Taylor, Kathy K GriendlingAbstract:Abstract —Recent evidence suggests that oxidative mechanisms may be involved in vascular smooth muscle cell (VSMC) hypertrophy. We previously showed that angiotensin II (Ang II) increases superoxide production by activating an NADH/NADPH oxidase, which contributes to hypertrophy. In this study, we determined whether Ang II stimulation of this oxidase results in H 2 O 2 production by studying the effects of Ang II on intracellular H 2 O 2 generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H 2 O 2 levels at 4 hours. Neither superoxide dismutase activity nor catalase activity was affected by Ang II; the SOD present in VSMCs is sufficient to metabolize Ang II–stimulated superoxide to H 2 O 2 , which accumulates more rapidly than it is degraded by catalase. This increase in H 2 O 2 was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT 1 receptor blocker losartan. In VSMCs stably transfected with antisense p22phox, a critical component of the NADH/NADPH oxidase in which oxidase activity was markedly reduced, Ang II–induced production of H 2 O 2 was almost completely inhibited, confirming that the source of Ang II–induced H 2 O 2 was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H 2 O 2 is a critical step in VSMC hypertrophy, a hallmark of many vascular diseases. Inhibition of intracellular superoxide dismutase by diethylthiocarbamate (1 mmol/L) also resulted in attenuation of Ang II–induced hypertrophy (62±2% inhibition). These data indicate that AT 1 receptor–mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H 2 O 2 , suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.
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angiotensin ii stimulates nadh and NADPH oxidase activity in cultured vascular smooth muscle cells
Circulation Research, 1994Co-Authors: Kathy K Griendling, C A Minieri, J D Ollerenshaw, R W AlexanderAbstract:The signaling pathways involved in the long-term metabolic effects of angiotensin II (Ang II) in vascular smooth muscle cells are incompletely understood but include the generation of molecules likely to affect oxidase activity. We examined the ability of Ang II to stimulate superoxide anion formation and investigated the identity of the oxidases responsible for its production. Treatment of vascular smooth muscle cells with Ang II for 4 to 6 hours caused a 2.7 +/- 0.4-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. This superoxide appeared to result from activation of both the NADPH and NADH oxidases. NADPH oxidase activity increased from 3.23 +/- 0.61 to 11.80 +/- 1.72 nmol O2-/min per milligram protein after 4 hours of Ang II, whereas NADH oxidase activity increased from 16.76 +/- 2.13 to 45.00 +/- 4.57 nmol O2-/min per milligram protein. The NADPH oxidase activity was stimulated by exogenous phosphatidic and arachidonic acids and was partially inhibited by the ...
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angiotensin ii stimulates nadh and NADPH oxidase activity in cultured vascular smooth muscle cells
Circulation Research, 1994Co-Authors: Kathy K Griendling, C A Minieri, J D Ollerenshaw, R W AlexanderAbstract:The signaling pathways involved in the long-term metabolic effects of angiotensin II (Ang II) in vascular smooth muscle cells are incompletely understood but include the generation of molecules likely to affect oxidase activity. We examined the ability of Ang II to stimulate superoxide anion formation and investigated the identity of the oxidases responsible for its production. Treatment of vascular smooth muscle cells with Ang II for 4 to 6 hours caused a 2.7 +/- 0.4-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. This superoxide appeared to result from activation of both the NADPH and NADH oxidases. NADPH oxidase activity increased from 3.23 +/- 0.61 to 11.80 +/- 1.72 nmol O2-/min per milligram protein after 4 hours of Ang II, whereas NADH oxidase activity increased from 16.76 +/- 2.13 to 45.00 +/- 4.57 nmol O2-/min per milligram protein. The NADPH oxidase activity was stimulated by exogenous phosphatidic and arachidonic acids and was partially inhibited by the specific inhibitor diphenylene iodinium. NADH oxidase activity was increased by arachidonic and linoleic acids, was insensitive to exogenous phosphatidic acid, and was inhibited by high concentrations of quinacrine. Both of these oxidases appear to reside in the plasma membrane, on the basis of migration of the activity after cellular fractionation and their apparent insensitivity to the mitochondrial poison KCN. These observations suggest that Ang II specifically activates enzyme systems that promote superoxide generation and raise the possibility that these pathways function as second messengers for long-term responses, such as hypertrophy or hyperplasia.
Vamsi K Mootha - One of the best experts on this subject based on the ideXlab platform.
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spatiotemporal compartmentalization of hepatic nadh and NADPH metabolism
Journal of Biological Chemistry, 2018Co-Authors: Vamsi K Mootha, Russell P Goodman, Sarah E CalvoAbstract:Compartmentalization is a fundamental design principle of eukaryotic metabolism. Here, we review the compartmentalization of NAD+/NADH and NADP+/NADPH with a focus on the liver, an organ that experiences the extremes of biochemical physiology each day. Historical studies of the liver, using classical biochemical fractionation and measurements of redox-coupled metabolites, have given rise to the prevailing view that mitochondrial NAD(H) pools tend to be oxidized and important for energy homeostasis, whereas cytosolic NADP(H) pools tend to be highly reduced for reductive biosynthesis. Despite this textbook view, many questions still remain as to the relative size of these subcellular pools and their redox ratios in different physiological states, and to what extent such redox ratios are simply indicators versus drivers of metabolism. By performing a bioinformatic survey, we find that the liver expresses 352 known or predicted enzymes composing the hepatic NAD(P)ome, i.e. the union of all predicted enzymes producing or consuming NADP(H) or NAD(H) or using them as a redox co-factor. Notably, less than half are predicted to be localized within the cytosol or mitochondria, and a very large fraction of these genes exhibit gene expression patterns that vary during the time of day or in response to fasting or feeding. A future challenge lies in applying emerging new genetic tools to measure and manipulate in vivo hepatic NADP(H) and NAD(H) with subcellular and temporal resolution. Insights from such fundamental studies will be crucial in deciphering the pathogenesis of very common diseases known to involve alterations in hepatic NAD(P)H, such as diabetes and fatty liver disease.
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genetically encoded tools for compartment specific manipulation of nad nadh or nadp NADPH ratios
Free Radical Biology and Medicine, 2016Co-Authors: Valentin Cracan, Denis V Titov, Zenon Grabarek, Vamsi K MoothaAbstract:Background The pool sizes and ratios of the pyridine nucleotides NAD(P)+ and NAD(P)H play a fundamental role in cellular metabolism and have emerged as key factors in numerous pathologies, including mitochondrial disorders, neurodegenerative diseases, cancer and aging. However, evaluating the impact of changes in concentrations of particular pyridine nucleotides in these conditions has not been possible due to lack of tools with which to directly perturb the NAD(P)+/NAD(P)H ratios. The aim of our study was to develop such tools, which are based on heterologous expression of naturally occurring or engineered water-forming NAD(P)H oxidases (NOXes). NOXes catalyze a four-electron reduction of oxygen to water using reducing equivalents of NAD(P)H, and their natural function is to detoxify O2 in several facultative or strictly anaerobic bacteria and parasitic protozoa. Methods Naturally occurring eukaryotic and bacterial NOXes were screened in human cells with or without a mitochondrial targeting sequence and assessed for their ability to consume oxygen and to change NAD(P)+/NAD(P)H ratios. The best candidates were overproduced in E.coli, purified and biochemically characterized for their substrate specificity and activity. Structural enzymology and rational mutagenesis were employed to engineer NAD(P)H specific variants. Results Our kinetic and structural studies revealed that bacterial NOX from Lactobacillus brevis (LbNOX) is strictly specific for NADH over NADPH, produces negligible H2O2, and has high catalytic activity compared to other candidate NOXes. Expression of LbNOX in mitochondria or cytoplasm of human cells is well tolerated and leads to a compartment-specific increase in the NAD+/NADH ratio. We used LbNOX to demonstrate the dependence of key metabolic fluxes and signaling on the cytosolic or mitochondrial NAD+/NADH ratios. In addition, LbNOX can fully complement electron transport chain function to support mammalian cell proliferation by re-oxidizing NADH to NAD+. Conclusions Taken together, the genetically encoded tools which we are developing provide a toolkit for compartment-specific manipulation of the NAD+/NADH and NADP+/NADPH ratios in living cells.
Charles B Kasper - One of the best experts on this subject based on the ideXlab platform.
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mechanistic studies on the reductive half reaction of NADPH cytochrome p450 oxidoreductase
Journal of Biological Chemistry, 1999Co-Authors: Anna L Shen, Charles B KasperAbstract:Abstract Site-directed mutagenesis has been employed to study the mechanism of hydride transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specifically, Ser457, Asp675, and Cys630 have been selected because of their proximity to the isoalloxazine ring of FAD. Substitution of Asp675 with asparagine or valine decreased cytochromec reductase activities 17- and 677-fold, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytochromec reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A., and Kasper, C. B. (1996) Biochemistry 35, 9451–9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly multiplicative decreases in cytochrome c reductase activity (774- and 22000-fold, respectively) with corresponding decreases in the rates of flavin reduction. For each mutation, increases were observed in the magnitudes of the primary deuterium isotope effects with NADPD, consistent with decreased rates of hydride transfer from NADPH to FAD and an increase in the relative rate limitation of hydride transfer. Asp675substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shifted the pK a of an ionizable group previously identified as necessary for catalysis (Sem, D. S., and Kasper, C. B. (1993)Biochemistry 32, 11539–11547) from 6.9 to 7.8. These results are consistent with a model in which Ser457, Asp675, and Cys630 stabilize the transition state for hydride transfer. Ser457 and Asp675interact to stabilize both the transition state and the FAD semiquinone, while Cys630 interacts with the nicotinamide ring and the fully reduced FAD, functioning as a proton donor/acceptor to FAD.
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interaction with arginine 597 of NADPH cytochrome p 450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and nadh
Biochemistry, 1993Co-Authors: Daniel S Sem, Charles B KasperAbstract:Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)
