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PubMed2005

Crystal structure of NAD-dependent malate dehydrogenase complexed with NADP(H).

For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme.

Binding Sites↗
PubMed2005

Zinc ions selectively inhibit steps associated with binding and release of NADP(H) during turnover of proton-translocating transhydrogenase.

Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. In membrane vesicles from Escherichia coli and Rhodospirillum rubrum, the transhydrogenase reaction (measured in the direction driving inward proton translocation) was inhibited by Zn(2+) and Cd(2+). However, depending on pH, the metal ions either had no effect on, or stimulated, "cyclic" transhydrogenation. They must, therefore, interfere specifically with steps involving binding/release of NADP(+)/NADPH: the steps thought to be associated with proton translocation. It is suggested that Zn(2+) and Cd(2+) bind in the proton-transfer pathway and block inter-conversion of states responsible for changing NADP(+)/NADPH binding energy.

Ion Transport↗
PubMed1999

Evidence for the stabilization of NADPH relative to NADP(+) on the dIII components of proton-translocating transhydrogenases from Homo sapiens and from Rhodospirillum rubrum by measurement of tryptophan fluorescence.

A unique Trp residue in the recombinant dIII component of transhydrogenase from human heart mitochondria (hsdIII), and an equivalent Trp engineered into the dIII component of Rhodospirillum rubrum transhydrogenase (rrdIII.D155W), are more fluorescent when NADP(+) is bound to the proteins, than when NADPH is bound. We have used this to determine the occupancy of the binding site during transhydrogenation reactions catalysed by mixtures of recombinant dI from the R. rubrum enzyme and either hsdIII or rrdIII.D155W. The standard redox potential of NADP(+)/NADPH bound to the dIII proteins is some 60-70 mV higher than that in free solution. This results in favoured reduction of NADP(+) by NADH at the catalytic site, and supports the view that changes in affinity at the nucleotide-binding site of dIII are central to the mechanism by which transhydrogenase is coupled to proton translocation across the membrane.

Animals↗
PubMed2002

Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum.

Clostridium acetobutylicum gapN was cloned and expressed in Escherichia coli BL-21. The IPTG-induced nonphosphorylating NADP-dependent GAPDH (GAPN) has been purified about 34-fold from E. coli cells and its physical and kinetic properties were investigated. The purification method consisted of a rapid and straightforward procedure involving anion-exchange and hydroxyapatite chromatographies. The purified protein is an homotetrameric of 204kDa exhibiting absolute specificity for NADP. Chromatofocusing analysis showed the presence of only one acidic GAPN isoform with an acid isoelectric point of 4.2. The optimum pH of purified enzyme was 8.2. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65 degrees C with activation energy of 18KJmol(-1). The apparent K(m) values for NADP and D-glyceraldehyde-3-phosphate (D-G3P) or DL-G3P were estimated to be 0.200+/-0.05 and 0.545+/-0.1 mM, respectively. No inhibition was observed with L-D3P. The V(max) of the purified protein was estimated to be 78.8 U mg(-1). The Cl. acetobutylicum GAPN was markedly inhibited by sulfhydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfhydryl groups in the catalytic activity.

Catalysis↗
PubMed2001

Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E. coli transhydrogenase.

The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266. A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values. It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.

Amino Acid Sequence↗
PubMed2002

Small changes in the activity of chloroplastic NADP(+)-dependent ferredoxin oxidoreductase lead to impaired plant growth and restrict photosynthetic activity of transgenic tobacco plants.

A ferredoxin-NADP+ oxidoreductase (FNR) cDNA from tobacco (Nicotiana tabacum cv. Samsun) was cloned and sequenced. Comparison of the deduced amino acid sequence revealed high identity to FNR proteins from Capsicum annuum, Pisum sativum, Spinacia oleracea and Vicia faba. Transgenic tobacco plants were generated that constitutively express the FNR cDNA in reverse orientation between the CaMV 35S promoter and the polyadenylation signal of the octopine synthase gene. Plants expressing the FNR antisense gene showed lower levels of FNR mRNA and protein accumulation, which was paralleled by a decrease in FNR activity. As a consequence, NADPH levels declined whereas NADP+ levels increased, leading to an unaltered NADP(H) pool. Growth rates, chlorophyll content and net CO2 uptake rates at high and low irradiances were strongly reduced in FNR antisense tobacco plants. These changes were accompanied by an over-reduced state of P700 as estimated by absorption changes at 820 nm. FNR control coefficients determined for the photosynthetic rate at saturating (C(R) = 0.94) and limiting (C(R) = 0.70) light conditions revealed a prominent role of this reductase in the regulation of photosynthesis.

Blotting, Western↗
PubMed2002

Probing the role of glutamic acid 139 of Anabaena ferredoxin-NADP+ reductase in the interaction with substrates.

The role of the negative charge of the E139 side-chain of Anabaena Ferredoxin-NADP+ reductase (FNR) in steering appropriate docking with its substrates ferredoxin, flavodoxin and NADP+/H, that leads to efficient electron transfer (ET) is analysed by characterization of several E139 FNR mutants. Replacement of E139 affects the interaction with the different FNR substrates in very different ways. Thus, while E139 does not appear to be involved in the processes of binding and ET between FNR and NADP+/H, the nature and the conformation of the residue at position 139 of Anabaena FNR modulates the precise enzyme interaction with the protein carriers ferredoxin (Fd) and flavodoxin (Fld). Introduction of the shorter aspartic acid side-chain at position 139 produces an enzyme that interacts more weakly with both ET proteins. Moreover, the removal of the charge, as in the E139Q mutant, or the charge-reversal mutation, as in E139K FNR, apparently enhances additional interaction modes of the enzyme with Fd, and reduces the possible orientations with Fld to more productive and stronger ones. Hence, removal of the negative charge at position 139 of Anabaena FNR produces a deleterious effect in its ET reactions with Fd whereas it appears to enhance the ET processes with Fld. Significantly, a large structural variation is observed for the E139 side-chain conformer in different FNR structures, including the E139K mutant. In this case, a positive potential region replaces a negative one in the wild-type enzyme. Our observations further confirm the contribution of both attractive and repulsive interactions in achieving the optimal orientation for efficient ET between FNR and its protein carriers.

Anabaena↗
PubMed2003

Open questions in ferredoxin-NADP+ reductase catalytic mechanism.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.

Bacterial Proteins↗
PubMed2000

Inhibition of the thioredoxin-dependent activation of the NADP-malate dehydrogenase and cofactor specificity.

The chloroplastic NADP-malate dehydrogenase is activated by reduction of its N- and C-terminal disulfides by reduced thioredoxin. The activation is inhibited by NADP(+), the oxidized form of the cofactor. Previous studies suggested that the C-terminal disulfide was involved in this process. Recent structural data pointed toward a possible direct interaction between the C terminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibition of activation has been investigated by changing the cofactor specificity of the enzyme by substitution of selected residues of the cofactor-binding site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofactor and support the idea of a direct interaction between the negatively charged C-terminal end of the enzyme and the positively charged nicotinamide ring of the cofactor, in agreement with the structural data. The structural requirements for cofactor specificity are modeled and discussed.

Amino Acid Sequence↗
PubMed2000

The role of active site arginines of sorghum NADP-malate dehydrogenase in thioredoxin-dependent activation and activity.

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.

Adenosine Monophosphate↗
PubMed2000

The NADP(H)-binding component (dIII) of human heart transhydrogenase: crystallization and preliminary crystallographic analysis.

Transhydrogenase is a membrane protein which uses the energy of the proton motive force to drive the reduction of NADP(+) by NADH. The enzyme has three domains: dII spans the membrane, while dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively. DIII from human heart transhydrogenase has been expressed in Escherichia coli. The purified protein has been crystallized with bound NADP(+) using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant. The crystals belong to the tetragonal space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 58.1, c = 251.0 A. A 2.1 A resolution native data set has been collected with an R(merge) of 6. 8%.

Binding Sites↗
PubMed1992

A mutation at Gly314 of the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change.

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the beta subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the beta subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314----Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314----Glu mutation. It is concluded that the mutation in the beta subunit abolished the NADP(H)-induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal beta subunit at Arg265 by trypsin. The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647-653].

Amino Acid Sequence↗
PubMed1996

Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP+ photoreduction.

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH--cytochrome c reductase reaction catalysed by ferredoxin--NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-->Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I. Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.

Base Sequence↗
PubMed1988

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase.

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.

Algorithms↗
PubMedDate not supplied

[Inhibition of adrenodoxin reductase by NADP+ and NADPH].

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.

Adrenal Cortex↗
PubMed1997

NADP-dependent enzymes. II: Evolution of the mono- and dinucleotide binding domains.

Nicotinamide adenine dinucleotides [NAD and NADP with both referred to as NAD(P)] are among the more diffuse redox cofactors. Despite their stereochemical similarity where the only difference is a phosphomonoester on the ribose near the adenine of NADP, they show different biochemical reactivities with NAD behaving as an oxidant and NADP as a reductant. NAD(P)-dependent enzymes generally share a common open alpha/beta fold with few exceptions only recently structurally characterized. This study of the molecular evolution of the NAD(P) binding domains, possible given the large number of known molecular structures, addresses two main questions: 1) can a common fold exist in different biological systems (divergent evolution) and 2) does a relationship exist among similar biological systems that display different folds (convergent evolution)? Both the structures of mono- and dinucleotide binding domains have been classified by cluster analysis based on the similarity evaluated by their main chain C alpha superposition. Moreover, the cofactor conformations and the stereochemical characteristics of their pockets have also been classified by analogous methods on the basis of the published tertiary structures. Two primary results appear: 1) the classification of the mononucleotide binding domains is different from that of the dinucleotide binding folds and 2) both divergent and convergent evolutionary pathways can be hypothesized, the latter less frequently observed and less pronounced but nevertheless evident. The generally accepted hypothesis that dinucleotide binding domains have evolved by gene duplication of primordial genes coding for the smaller mononucleotide binding domains is acceptable but the two halves of the resulting dinucleotide binding domains are evolutionarily uncorrelated. The NH2-terminal mononucleotide binding domain is less variable than the COOH-terminal half, probably because it involves the binding of the ADP moiety of NAD(P) invariant in all examined systems. There is evidence to postulate that evolutionary pathways for NAD(P)-dependent enzymes are both divergent and convergent. In fact, nearly all combinations of similarity dissimilarity in overall fold, cofactor conformation, and cofactor binding pocket structural characteristics for each enzyme pair examined are possible. The NAD(P)-dependent enzymes apparently provide a canonical example of an evolutionary principle that "anything goes."

Animals↗
PubMed1979

Abnormal erythrocyte metabolism in hepatic disease: effect of NADP repletion.

Erythrocytes from ten patients with severe liver disease displayed low methylene blue-stimulated hexose monophosphate (HMP) shunt activity and glucose recycling despite elevated total glucose consumption when compared to controls. Heinz body formation was increased and reduced glutathione concentration significantly decreased. After hemolysis, no differences in methylene-blue estimulated HMP shunt activity or glucose recycling could be demonstrated between patients and controls. The addition of 2- and 4-mM NADP to the hemolysates produced significantly greater HMP shunt activity and glucose recycling in the patients' hemolysates. The addition of NADPH to the incubation mixture produced no significant stimulation of either HMP shunt activity or glucose recycling, unless methylene blue was also added. Omission of NAD or phosphate from the incubation mixture produced no change in shunt metabolism. The absence of supplemental ATP resulted in extremely low shunt metabolism and refractoriness to NADP stimulation in both patients and controls. In the absence of additional magnesium, a reduction of shunt metabolism was noted. These data suggest that the defect in stimulated shunt metabolism in the intact erythrocytes of patients with hepatic disease does not result from an absolute enzyme deficiency, but rather from an unavailability of NADP or other cofactor.

Adult↗
PubMed1986

G-6-PD Walter Reed: possible insight into "structural" NADP in G-6-PD.

A new G-6-PD variant, G-6-PD Walter Reed, causing hereditary nonspherocytic hemolytic anemia is characterized. This variant is unusual in that its stability requires the presence of high concentrations of NADP, while its Km for NADP is normal. This finding is consistent with the suggestion that G-6-PD has two separate binding sites, a high affinity "structural" site and a lower affinity catalytic site. The mutation in G-6-PD Walter Reed, like that of the previously described variant, G-6-PD Torrance, may be due to a mutation of the "structural" site for NADP.

Anemia, Hemolytic, Congenital↗