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PubMed2002

Intensity of free radical processes and regulation of cytoplasmic NADP-isocitrate dehydrogenase in rat cardiomyocytes under normal and ischemic conditions.

The intensity of free radical processes and the regulation of NADP-isocitrate dehydrogenase (EC 1.1.1.42; NADP-IDH) activity have been studied in the cytoplasmic fraction of normal and ischemized rat myocardium. Chemiluminescence parameters, such as the light sum (S) of slow flash and the tangent of the kinetic curve slope angle (tanalpha1), which characterize the intensity of free radical processes, were increased in ischemia 2.1- and 20.0-fold, respectively. The slow flash intensity (Imax) was increased 22-fold. The contents of lipid peroxidation products--diene conjugates and malonic dialdehyde--were increased 11.9- and 4.7-fold, respectively, suggesting pronounced oxidative stress. Using homogenous enzyme preparations of NADP-IDH isolated from the normal and experimentally ischemized rat myocardium, a number of catalytic properties of the enzyme were characterized for normal and pathologic conditions. NADP-IDH from the normal and ischemized myocardium had the same electrophoretic mobility and was regulated similarly by Fe2+, Cu2+, Zn2+, and also with succinate and fumarate. However, under normal and pathologic conditions NADP-IDH was different in the affinity for substrates and in the sensitivity to inhibitory effects of hydrogen peroxide, reduced glutathione, and of Ca2+. The degree of synergy in the enzyme inhibition with Fe2+ and H2O2 was less pronounced in ischemia. The inhibitory effect of the reaction product 2-oxoglutarate was higher under normal conditions than in ischemia (the Ki values were 0.22 and 0.75 mM, respectively). The specific features of the NADP-IDH regulation in ischemia are suggested to promote the stimulation of the enzyme functioning during increased level of free radical processes, and this seems to be important for NADPH supplying for the glutathione reductase/glutathione peroxidase antioxidant system of cardiomyocytes.

Animals↗
PubMed2006

Identification of domains involved in tetramerization and malate inhibition of maize C4-NADP-malic enzyme.

C(4) photosynthetic NADP-malic enzyme (ME) has evolved from non-C(4) isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C(4) and non-C(4)-NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C(4)-NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C(4) isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C(4)-NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C(4) isoform in vivo, with the enzyme presenting maximum activity while photosynthesis is in progress.

Allosteric Site↗
PubMed1985

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L.

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K(+) and malate. DCMU inhibited the increase of K(+) and malate, and consequently swelling.Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

Journal Article↗
PubMed1990

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants.

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C(3) plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C(4) plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C(4) plants, 0.5% in the CAM plant, and less than 0.01% in C(3) plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C(3) and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C(3) and CAM plants than to those of C(4) plants.

Journal Article↗
PubMed1988

Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves.

The cytosolic isocitrate dehydrogenases (NADP-IDH) were purified to homogeneity from pea roots and green leaves with a high yield by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephacryl S-200 gel filtration, Matrex red-A affinity chromatography and phenyl-Superose HR 5/5 HPLC. Both isoenzymes were dimeric proteins, consisting of two apparently identical 41-kDa subunits, having similar secondary structures with an alpha-helical content of 20% and a beta-pleated sheet content of 43%. Similarly immunoassays suggested that the two isoenzymes were closely related in terms of antigenic determinants. However, the two proteins were distinguishable by their electrophoretic mobilities and amino acid compositions. The profiles of the two isoenzymes as a function of pH were similar and exhibited a broad pH optimum from 8.5 to 9.0 with Mg2+ as cofactor and 8.0 to 8.5 when Mn2+ was used. Compared to the root isoenzyme, the leaf NADP-IDH appeared to be more heat-labile. However, these isoenzymes exhibited similar behavior for thermal denaturation in the presence of bovine serum albumin and were stabilized upon addition of substrate, metal and coenzyme. Their values of activation energy were estimated as 47 kJ/mol. When using Mn2+ as cofactor, the two isoenzymes displayed identical Km(Mn2+), Km(DL-isocitrate) and Km(NADP) values, which were calculated to be 2.1 microM, 5.7 microM and 2.7 microM respectively. With Mg2+ as cofactor, their Km(Mg2+) K(DL-isocitrate)m and Km(NADP) values were also not statistically different, being 34.0 microM, 15.2 microM and 2.6 microM for the root NADP-IDH, and 29.0 microM, 20.3 microM and 3.1 microM for the leaf isoenzyme. From the above data it can be concluded that although the cytosolic NADP-IDH in pea roots and leaves are organ-specific isozymes, their similar physicochemical and kinetic properties suggest that the two isozymes might be involved in identical metabolic functions.

Amino Acids↗
PubMed2001

Establishment of a mass-production system for NADP using bacterial inorganic polyphosphate/ATP-NAD kinase.

The inorganic polyphosphate/ATP-dependent NAD kinase (Ppnk) of Mycobacterium tuberculosis was applied to the mass-production of NADP from NAD and inorganic polyphosphate (metaphosphate). When Ppnk purified from recombinant Escherichia coli cells overexpressing the M. tuberculosis Ppnk was used, 30 mM (27 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The recombinant E. coli cells were immobilized in polyacrylamide gel, and treated with acetone to render the cells permeable to substrates and products. When acetone-treated immobilized cells were used, 16 mM (14 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The isolation of NADP formed in the reaction mixture was easy because of the absence of by-products (ATP degradation compounds), and this NADP production system using purified Ppnk or immobilized recombinant E. coli cells expressing Ppnk is thought to be feasible in the production of NADP on an industrial scale.

Journal Article↗
PubMedDate not supplied

[Characteristics of NADP-dependent isocitrate dehydrogenase functioning in pea leaves during salt stress].

In pea seedlings transferred to a medium containing 3% NaCl, NADP-dependent isocitrate dehydrogenase (NADP-IDH) activity in leaves increased more than twofold during the first hour, decreased, and increased again (approximately 1.5 times) by the 14th hour of exposure. Some catalytic and regulatory properties of NADP-IDH were studied using enzyme preparations purified from normal plants and plants exposed to salt stress. The results showed that Km for NADP-IDH in reactions with isocitrate and NADP decreased under stress by factors of 2 and 3, respectively. Specific features of enzyme activity regulation by citrate and trans-aconitate under these conditions were revealed. The inhibitory effects of gamma-aminobutyric acid (GABA) and 2-oxoglutarate on NADP-IDH from plants exposed to salt stress was stronger than that on the enzyme from normal plants. Glutamine and glutamate slightly activated the enzyme in both cases.

Aconitic Acid↗
PubMed1988

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase.

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.

Amino Acid Sequence↗
PubMed2001

Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP.

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis. The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes. Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.

Amino Acid Sequence↗
PubMed2004

NADP-glutamate dehydrogenase activity is increased under hyperosmotic conditions in the halotolerant yeast Debaryomyces hansenii.

Glutamate plays an important role in osmoprotection in various bacteria. In these cases, increased intracellular glutamate pools are not attributable to the NADP-dependent glutamate dehydrogenase (NADP-GDH) or the glutamate synthase, which do not increase their activities under hyperosmotic conditions, but rather to changes in other enzymes involved in glutamate metabolism. We performed a study which indicates that, as opposed to what happens in bacteria, the activity of NADP-GDH is fivefold higher when the halotolerant yeast Debaryomyces hansenii is grown in the presence of 1 M NaCl, compared with growth in media with no added salt. Since purified NADP-GDH activity in vitro was not enhanced by the presence of salt and was more sensitive to ionic strength than the two isoenzymes from S. cerevisiae, increased enzyme synthesis is the most plausible mechanism to explain our results. We discuss the possibility that increased NADP-GDH activity in D. hansenii plays a role in counteracting the inhibitory effect of high ionic strength on the activity of this enzyme.

Ammonium Chloride↗
PubMed1994

Binding of the 2',5'-ADP subsite stimulates cyclohydrolase activity of human NADP(+)-dependent methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

The bifunctional dehydrogenase/cyclohydrolase domain of the human trifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase catalyzes two sequential reactions with significant channeling of the intermediate, methenyltetrahydrofolate. Equilibrium dialysis established that a single, high-affinity NADP+ binding site exists per monomer of the dimeric enzyme. Kinetic characterization of NADP+ binding to the dehydrogenase using analogs as inhibitors demonstrated that affinity for this substrate is due almost exclusively to binding at the 2',5'-ADP subsite. The same structural specificities for binding are exhibited by these analogs in their effects on the cyclohydrolase. Both NADP+ and its 3-aminopyridine analog AADP partially inhibit the activity of the cyclohydrolase when assayed with added methenyltetrahydrofolate as substrate. However, under the same conditions, the cyclohydrolase is actually activated by 2',5'-ADP; activation requires the presence of the 5'-phosphate since 2'-AMP binds but does not activate. Nicotinamide ribose monophosphate (NMN) has no detectable effect either alone or in combination with 2',5'-ADP. The results are consistent with the existence of a shared dehydrogenase/cyclohydrolase active site proximal to the 2',5'-ADP subsite. NADP+ reduces the rate of the fully activated cyclohydrolase by 2-fold. Inhibition appears to be due to the loosely bound nicotinamide ring interacting with the common folate subsite, resulting in only partial inhibition by NADP+. The interaction of 2',5'-ADP with the cyclohydrolase suggests a potential role for this portion of the molecule in promoting the efficiency of the channeling of endogenously generated methenyltetrahydrofolate.

Adenine Nucleotides↗
PubMed2005

Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR.

Ferredoxin-NADP(+) reductase (FNR) catalyzes the reduction of NADP(+) through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP(+)- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP(+) was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP(+), respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites.

Amino Acid Sequence↗
PubMed2005

C-terminal tyrosine of ferredoxin-NADP+ reductase in hydride transfer processes with NAD(P)+/H.

Ferredoxin-NADP+ reductase (FNR) catalyzes the reduction of NADP+ to NADPH in an overall reversible reaction, showing some differences in the mechanisms between cyanobacterial and higher plant FNRs. During hydride transfer it is proposed that the FNR C-terminal Tyr is displaced by the nicotinamide. Thus, this C-terminal Tyr might be involved not only in modulating the flavin redox properties, as already shown, but also in nicotinamide binding and hydride transfer. FNR variants from the cyanobacterium Anabaena in which the C-terminal Tyr has been replaced by Trp, Phe, or Ser have been produced. All FNR variants show enhanced NADP+ and NAD+ binding, especially Tyr303Ser, which correlates with a noticeable improvement of NADH-dependent reactions. Nevertheless, the Tyr303Ser variant shows a decrease in the steady-state kcat value with NADPH. Fast kinetic analysis of the hydride transfer shows that the low efficiency observed for this mutant FNR under steady-state conditions is not due to a lack of catalytic ability but rather to the strong enzyme-coenzyme interaction. Three-dimensional structures for Tyr303Ser and Tyr303Trp variants and its complexes with NADP+ show significant differences between plant and cyanobacterial FNRs. Our results suggest that modulation of coenzyme affinity is highly influenced by the strength of the C-terminus-FAD interaction and that subtle changes between plant and cyanobacterial structures are able to modify the energy of that interaction. Additionally, it is shown that the C-terminal Tyr of FNR lowers the affinity for NADP+/H to levels compatible with steady-state turnover during the catalytic cycle, but it is not involved in the hydride transfer itself.

Amino Acid Sequence↗
PubMed2004

Morphology-associated expression of NADP-dependent glutamate dehydrogenases during yeast-mycelium transition of a dimorphic fungus Benjaminiella poitrasii.

Benjaminiella poitrasii, a dimorphic zygomycetous fungus possesses three glutamate dehydrogenases, one requiring NAD while the other two use NADP as a coenzyme. In the activity staining after electrophoresis on native polyacrylamide gel NAD- dependent glutamate dehydrogenase revealed the presence of one enzyme that was expressed in both, yeast- and mycelium-form cells. While in case of NADP- dependent glutamate dehydrogenase two distinct activity bands that were differentially expressed in yeast- and mycelium-form cells were seen. Interestingly, during yeast-mycelium transition and reverse, quantitative changes in form-specific native NADP-dependent glutamate dehydrogenase activities were seen. The biochemical data on temperature and pH optima, thermostability, and kinetic properties confirmed the presence of two NADP-dependent proteins in B. poitrasii, parent strain. The monomorphic mutant (Y-5, yeast form) showed NADP- glutamate dehydrogenase similar to parent yeast-form enzyme. For the first time the significance of differential expression of these enzymes during morphological transition in B. poitrasii has been suggested.

Enzyme Stability↗
PubMed2003

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.

Adrenodoxin↗
PubMed1999

Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. A study of structure and function.

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H). A model defining betaA, alphaB, betaB, betaD, and betaE of this domain is presented. To test this model, four single cysteine mutants (cfbetaA348C, cfbetaA390C, cfbetaK424C, and cfbetaR425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, beta-Ala348, beta-Arg350, beta-Ala390, beta-Asp392, and beta-Lys424 are located in or close to the NADP(H) site. More specifically, beta-Ala348 succeeds betaB. The remarkable reactivity of betaR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). beta-Ala390 and beta-Asp392 terminate or succeed betaD, and are thus, together with the region following betaA, creating the switch point crevice where NADP(H) binds. beta-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase.

Amino Acid Sequence↗
PubMed2003

Interactions between transhydrogenase and thio-nicotinamide Analogues of NAD(H) and NADP(H) underline the importance of nucleotide conformational changes in coupling to proton translocation.

Transhydrogenase couples the reduction of NADP+ by NADH to inward proton translocation across mitochondrial and bacterial membranes. The coupling reactions occur within the protein by long distance conformational changes. In intact transhydrogenase and in complexes formed from the isolated, nucleotide-binding components, thio-NADP(H) is a good analogue for NADP(H), but thio-NAD(H) is a poor analogue for NAD(H). Crystal structures of the nucleotide-binding components show that the twists of the 3-carbothiamide groups of thio-NADP+ and of thio-NAD+ (relative to the planes of the pyridine rings), which are defined by the dihedral, Xam, are altered relative to the twists of the 3-carboxamide groups of the physiological nucleotides. The finding that thio-NADP+ is a good substrate despite an increased Xam value shows that approach of the NADH prior to hydride transfer is not obstructed by the S atom in the analogue. That thio-NAD(H) is a poor substrate appears to be the result of failure in the conformational change that establishes the ground state for hydride transfer. This might be a consequence of restricted rotation of the 3-carbothiamide group during the conformational change.

Binding Sites↗
PubMed2006

Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange.

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

Chloroplasts↗