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At least 55 records · Page 3Linked to original sources
PubMed1980

Reconstitution of a formate-NADP+ oxidoreductase from formate dehydrogenase and a 5-deazaflavin-linked NADP+ reductase isolated from Methanococcus vannielii.

The formate-dependent reduction of NADP+ by extracts of Methanococcus vannielii is catalyzed by a coupled system consisting of formate dehydrogenase, a 5-deazaflavin cofactor, and 5-deazaflavin-dependent NADP+ reductase. All three components were purified from crude extracts of M. vannielii. Recombination of these components reconstituted the formate-NADP+ oxidoreductase system. The formate dehydrogenase also can utilize FAD, FMN, and a number of artificial dyes as electron acceptors, but these do not replace the 5-deazaflavin cofactor in the coupled enzyme system. The reduced form of 5-deazaflavin binds readily to the NADP+ reductase apoprotein and is not dissociated by ammonium sulfate treatment at neutral pH under anaerobic conditions. This electron transfer cofactor from M. vannielii is identical in many of its properties to the 5-deazaflavin isolated from other methane-producing bacteria.

Aldehyde Oxidoreductases↗
PubMed1999

A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.

Amino Acid Sequence↗
PubMed1975

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.

Alkaline Phosphatase↗
PubMed1997

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase.

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO2 reduction to CH4 contain either an NADP-dependent or a coenzyme F420-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N5, N10-methylenetetrahydromethanopterin dehydrogenase and the N5, N10-methylenetetrahydromethanopterin reductase, two enzymes involved in CO2 reduction to CH4, are specific for F420. This raised the question how F420H2 is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F420 with NADPH. The F420-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced.

Alcohol Oxidoreductases↗
PubMed2002

The flavoenzyme ferredoxin (flavodoxin)-NADP(H) reductase modulates NADP(H) homeostasis during the soxRS response of Escherichia coli.

Escherichia coli cells from strain fpr, deficient in the soxRS-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV). Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in fpr cells exposed to MV. FPR inactivation did not affect the MV-driven soxRS response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool. Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on soxRS induction and failed to protect fpr cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria.

Bacterial Proteins↗
PubMedDate not supplied

[Isolation of highly-purified NADP(H)-dependent enzymes from the rat liver using NADP-hydrazidoadipoyl oxypropyl sepharose].

NADP-hydrazidoadipoyl oxypropyl sepharose was synthesized from epoxyactivated sepharose through a hydrazid derivative and used for isolation of NADP(H)-dependent enzymes such as glutathione reductase, isocitrate dehydrogenase and malate dehydrogenase. The isolation technique involves fractionation with ammonium sulphate, affinity chromatography on NADP-hydrazidoadipoyl oxypropyl sepharose and chromatography on hydroxylapatite. The proposed technique enabled the authors to obtain malate dehydrogenase isocitrate dehydrogenase and glutathione reductase preparations homogeneous according to SDS-electrophoresis in polyacrylamide gel.

Animals↗
PubMed1998

Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal NADP+ binding and electron transfer.

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg100 and Arg264 may be directly involved in the proper NADP+/NADPH orientation for an efficient electron-transfer reaction. Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119 FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP+ and to ferredoxin/flavodoxin. Arg100 has been replaced with an alanine, which removes the positive charge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutation has been made at Arg264 by replacing it with a glutamic acid. Results with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Both mutants have been kinetically characterized by steady-state as well as fast transient kinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved. The results reported herein reveal important conceptual information about the interaction of FNR with its substrates. A critical role is confirmed for the long, positively charged side chain of Arg100. Studies on the Arg264Glu FNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or for nicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behavior in its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin.

Alanine↗
PubMed2005

A novel NADPH:(bound) NADP+ reductase and NADH:(bound) NADP+ transhydrogenase function in bovine liver catalase.

Many catalases have the shared property of containing bound NADPH and being susceptible to inactivation by their own substrate, H2O2. The presence of additional (unbound) NADPH effectively prevents bovine liver and human erythrocytic catalase from becoming compound II, the reversibly inactivated state of catalase, and NADP+ is known to be generated in the process. The function of the bound NADPH, which is tightly bound in bovine liver catalase, has been unknown. The present study with bovine liver catalase and [14C]NADPH and [14C]NADH revealed that unbound NADPH or NADH are substrates for an internal reductase and transhydrogenase reaction respectively; the unbound NADPH or NADH cause tightly bound NADP+ to become NADPH without becoming tightly bound themselves. This and other results provide insight into the function of tightly bound NADPH.

Animals↗
PubMed1984

Binding of NAD and NADP dimers to NAD- and NADP-dependent dehydrogenases.

Interaction of the electrolytically prepared dimers of nicotinamide adenine nucleotide, (NAD)2, and nicotinamide adenine nucleotide phosphate, (NADP)2, with lactate, alcohol, glyceraldehyde 3-phosphate, alpha-glycerophosphate, glutamate and glucose-6-phosphate dehydrogenase has been studied using the quenching of protein fluorescence, kinetics of inhibition and the stopped-flow method. It has been shown that these enzymes are able to bind dimers preserving their coenzyme specificity. The most efficient binding of (NAD)2 has been observed in the case of glutamate and lactate (bovine heart) dehydrogenase, the dissociation constants being 6 and 8 microM, respectively. (NADP)2 affinity to glutamate and glucose-6-phosphate dehydrogenase is also fairly high. More detailed studies on the interactions of dimers with alcohol and glutamate dehydrogenase have shown that the binding to the coenzyme binding site is the prerequisite for the association. However, some additional stabilizing interactions with other enzyme groups are not excluded, though (NAD)2 does not bind to the known binding sites of these enzymes, such as the substrate pocket of alcohol dehydrogenase and the regulatory binding sites for ADP and GTP of glutamate dehydrogenase.

Adenosine Diphosphate↗