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PubMed1997

Gene analysis of an NADP-linked isocitrate dehydrogenase localized in peroxisomes of the n-alkane-assimilating yeast Candida tropicalis.

In n-alkane-utilizing yeast, Candida tropicalis, two NADP-linked isocitrate dehydrogenase (NADP-IDH) isozymes are present, one in mitochondria (Mt-NADP-IDH) and the other in peroxisomes (Ps-NADP-IDH). Here we report the isolation, sequencing, and expression of the gene encoding Ps-NADP-IDH (CtIDP2), distinct from the Mt-NADP-IDH gene (CtIDP1). Based on the N-terminal amino acid sequence of purified Ps-NADP-IDH, a cDNA fragment specific for Ps-NADP-IDH was obtained by the 5'-RACE method. Using this fragment as a probe, the genomic CtIDP2 gene was isolated. Nucleotide sequence analysis of CtIDP2 disclosed that the region encoding CtIdp2p had a length of 1233 bp, corresponding to 411 amino acid residues. The deduced N-terminal amino acid sequence matched the results obtained from the purified protein. When this CtIDP2 was expressed in Saccharomyces cerevisiae using the C. tropicalis isocitrate lyase gene promoter (UPR-ICL), high intracellular NADP-IDH activity was observed. Comparison of amino acid sequences and phylogenetic tree analysis with NADP-IDH enzymes from all reported eukaryotic sources revealed that mammalian mitochondrial NADP-IDHs formed a cluster, as did plant NADP-IDHs. CtIdp2p and other yeast NADP-IDHs were not included in these clusters and seemed to diverge at an early stage from all other enzymes of higher eukaryotes. Ps-NADP-IDH had no typical C-terminal peroxisomal targeting signal and no processing was demonstrated at the N-terminus. However, we could find a region near the N-terminus of the protein with high similarity to both the putative N-terminal peroxisomal targeting signal sequence of Fox3p of S. cerevisiae and an internal region of Pox4p of C. tropicalis. The results of northern blot analysis indicated that the biosynthesis of CtIdp2p was induced in a medium containing alkanes as a carbon source, where profuse proliferation of peroxisomes is observed.

Alkanes↗
PubMed2004

Maize recombinant non-C4 NADP-malic enzyme: a novel dimeric malic enzyme with high specific activity.

Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C(4)-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C( 4) NADP-ME. Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed in vivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2 gene and the likely precursor to the evolution of the photosynthetic C(4) NADP-ME) and the 62 kDa isoform (implicated in C(4) photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C(4) isoenzyme in maize.

Amino Acid Sequence↗
PubMed1983

Light-stimulated synthesis of NADP malic enzyme in leaves of maize.

Illumination of etiolated maize plants for 80 h brings about a 15-20-fold increase in activity of NADP malic enzyme (EC 1.1.1.40). Increases in NADP malic enzyme protein and in the level of translatable mRNA for this protein occur simultaneously with the activity increase. Radiolabeled amino acids are also incorporated into NADP malic enzyme during this time. These results are consistent with the conclusion that an increase in NADP malic enzyme activity during greening results from de novo synthesis of NADP malic enzyme protein. Polyadenylated RNA extracted from greening maize leaves directs the synthesis in vitro of a protein 12,000 daltons larger than NADP malic enzyme purified from corn leaves. This protein is a precursor of NADP malic enzyme because 1) both the precursor and mature NADP malic enzyme are immunoprecipitated by antibody made against NADP malic enzyme purified from corn leaves, 2) both NADP malic enzyme protein and the level of mRNA for the precursor increase during greening, and 3) peptide maps of the precursor and of mature NADP malic enzyme are very similar. Mature NADP malic enzyme and its precursor (synthesized in vitro) both migrate on sodium dodecyl sulfate-polyacrylamide gradient gels as doublet bands. Peptide analyses show all bands to be structurally related.

Kinetics↗
PubMed1985

Phosphorus-31 nuclear magnetic resonance and electronic spectroscopic studies of adrenodoxin reductase and its binary complex with NADP+.

The 31P NMR spectra of NADPH-adrenodoxin reductase and its complex with NADP+ are reported. The spectrum of adrenodoxin reductase showed two doublets arising from the phosphorus nuclei in the pyrophosphate group of FAD. Both doublets were shifted upfield to different extents in comparison with those of free FAD. Further, one of the doublets of phosphorus nuclei of the pyrophosphate group of bound NADP+ in the complex of adrenodoxin reductase and NADP+ was considerably shifted upfield in comparison with that of free NADP+. The spectrum of the complex of the reductase and NADP+ showed that the resonance of the 2'-phosphate group of NADP+ bound to the reductase was shifted downfield by 1.37 ppm compared with that of free NADP+ in the dianionic state. The 2'-phosphate resonance of bound NADP+ was independent of pH within the physiological range, whereas that of free NADP+ changed according to its ionization. The resonance of the 2'-phosphate group of NADP+ bound to the reductase also revealed that the ratio for the complex of NADP+ and the reductase was 1:1, and that this complex formation was inhibited by a high KCl concentration. These results were confirmed by electronic spectroscopic studies.

Adrenal Cortex↗
PubMed1997

NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding.

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moieties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design.

Animals↗
PubMed1997

NADP-malic enzyme from the C4 plant Flaveria bidentis: nucleotide substrate specificity.

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) was purified to near-homogeneity from leaves of the C4 dicot Flaveria bidentis and shown to possess intrinsic NAD-dependent malic enzyme activity. The NAD-dependent activity is optimal at pH 7.5 and in the presence of Mn2+. The Km for NAD is very high (20 mM), while the Vmax is 50% greater than the Vmax with NADP under the same conditions. The NAD-dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (Ki approximately 2 microM). This very low Ki reflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, the Km for NADP is 1.5 microM while the Ki for NADPH is 2 microM. Chicken liver NADP-ME also has NAD-dependent activity that is inhibited by low concentrations of NADPH. These results indicate that the NAD- and NADP-dependent activities are likely catalyzed by the same active site. The use of NAD as an alternative coenzyme revealed interactions between the binding of coenzyme and metal ions on the Km values of each of the other participants in the malic enzyme reaction. Thus, the affinity of malic enzyme for the divalent metal ions Mg2+ and particularly Mn2+ as well as the other substrate L-malate is also dependent on the nucleotide coenzyme substrate. In turn, the divalent metal ion influences the affinity of the enzyme for the coenzyme as well as L-malate. With NADP as substrate the Km for Mn2+ is 4 microM, whereas with NAD the Km is 300 microM. The relatively high affinity of the enzyme for Mn2+ and low affinity for NAD required the use of metal ion buffers when determining these values because of the substantial depletion of free Mn2+ caused by binding to NADP.

Animals↗
PubMed1997

A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+.

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP. In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs. In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G). Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%). The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form. In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32. Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains. In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.

Binding Sites↗
PubMed1998

Thio-NADP is not an antagonist of NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca2+ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995] J. Biol. Chem. 270, 3216-3223). Its effects in sea-urchin egg homogenates were investigated. At 50 microM, thio-NADP activates partial Ca2+ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca2+ releasing activity of 50 microM thio-NADP and reduces the subsequent inhibition by 73.7 +/- 1.3%. The residual inhibitory effect is no more than that exerted by 50 microM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by 32P-NAADP binding studies. Unpurified thio-NADP inhibits the specific 32P-NAADP binding to egg microsomes with an IC50 of 40 microM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 microM, similar to the extent of inhibition effected by 40 microM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca2+ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca2+ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.

Adenosine Diphosphate Ribose↗
PubMed1994

Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+.

We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp-39 is replaced with asparagine, has a 60-fold increase in affinity for NADP+ and a 1.5-fold increase in kcat compared to wild-type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263-267] and proposed that this part of ADH is close to the 2'-phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala-46 with an argine residue, and A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three-dimensional structure of the NAD(+)-binding region of ADH. The A46R mutant has a 2.5-fold lower Km(app)NADP+ and a 3-fold higher kcat with NADP+ compared to wild-type ADH; binding of NAD+ to the mutant was unchanged and kcat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of kcat/Km of NAD+ to NADP+ is 85, over ten-fold lower than that for wild-type ADH. Our model of the 3D structure of the NAD(+)-binding region of ADH shows that Ala-46 is over 10 A from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp-39 and the 2'-phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30-fold lower Km(app)NADP+ and 8-fold higher kcat with NADP+ as a cofactor compared to wild-type ADH; binding of NAD+ to this double mutant was lowered by 5-fold and kcat was increased by 1.5-fold. As a result, kcat/Km for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; kcat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.

Alanine↗
PubMed2000

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme.

NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C4 species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C4 subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts.

Chlorophyll↗
PubMed1980

Study of the interaction between NADP-dependent dehydrogenase and immobilized adenosine 2'-monophosphate by means of affinity electrophoresis.

A water-soluble 8-substituted adenosine 2'-monophosphate-polyacrylamide (8-sub-2'-AMP-PA) was prepared as a new affinity ligand for the determination of the dissociation constants of the interactions between immobilized 8-sub-2'-AMP and NADP-dependent dehydrogenases (NADP-dependent DH), NAD-dependent dehydrogenases (NAD-dependent DH) and phosphorylase by means of affinity electrophoresis. From the dissociation constants, it was found that NADP-dependent DH had a much stronger affinity to immobilized 8-sub-2'-AMP than did NAD-dependent DH and phosphorylase. On the other hand, NADP-dependent DH had a much weaker affinity to immobilized 8-sub-5'-AMP than did NAD-dependent DH. The effects of NADP+ and NAD+ on the interaction between immobilized 8-sub-2'-AMP and NADP-dependent DH were also investigated by means of affinity electrophoresis. NADP+ inhibited the interaction specifically, but NAD+ did not inhibit the interaction. These results indicate that 8-sub-2'-AMP binds to the coenzyme binding site of NADP-dependent DH, while other compounds, such as 8-sub-5'-AMP and NAD+, do not bind to NADP-depenndent DH. Such a difference suggests that the phosphate group at position 2' in 2'-AMP and NADP+ is important for the binding at the coenzyme binding site of NADP-dependent DH.

Adenosine Monophosphate↗
PubMed2003

Maize C4 NADP-malic enzyme. Expression in Escherichia coli and characterization of site-directed mutants at the putative nucleoside-binding sites.

Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO(2), and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C(4)-specific NADP-ME has evolved from C(3)-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C(4)-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and K(m) for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the k(cat) was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C(4) NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.

Amino Acids↗
PubMed1991

Purification and properties of F420- and NADP(+)-dependent alcohol dehydrogenases of Methanogenium liminatans and Methanobacterium palustre, specific for secondary alcohols.

The F420-dependent alcohol dehydrogenase (ADH) of Methanogenium liminatans and the NADP(+)-dependent ADH of Methanobacterium palustre were purified to homogeneity. The native F420-dependent ADH of Mg. liminatans had a molecular mass of 150 kDa and consisted of four (presumably identical) subunits with a mass of 39 kDa. The temperature optimum was 42 degrees C, the optimum pH 6.0 and NaCl or KCl were inhibitory. The NADP(+)-dependent ADH of Mb. palustre had a molecular mass of 175 kDa and consisted also of four (presumably identical) subunits with a mass of 44 kDa. The temperature optimum was 60 degrees C, the optimum pH 8.0 and optimal activity was observed in the presence of 500 mM NaCl or KCl. The ADHs of both organisms catalysed the oxidation of various secondary and cyclic alcohols to the corresponding ketones and the reverse reaction. No primary alcohols were apparently oxidized. The NADP(+)-dependent ADH of Mb. palustre contained 4-8 mol atoms zinc/mol enzyme and was inhibited by low concentrations of iodoacetate and 4-hydroxymercuribenzoate, whereas the F420-dependent ADH of Mg. liminatans presumably contained no zinc ions and was inhibited by 1,10-phenanthroline or high concentrations (e.g. 100 microM) of 4-hydroxymercuribenzoate. Polyclonal antibodies against the NADP(+)-dependent ADH of Mb. palustre precipitated only the homologous ADH. A precipitation of the NADP(+)-dependent ADH of Methanocorpusculum parvum required a 10-fold higher antibody concentration, showing at least a distant relationship of both ADHs. Antibodies against the NADP(+)-dependent ADH of Mcp. parvum, however, formed precipitates with the homologous ADH of Mcp. parvum and with the NADP(+)-dependent ADH of Mb. palustre. They also formed precipitates with the ADH of Thermoanaerobium brockii, which is not related to methane bacteria. Antibodies against the F420-dependent ADH of Mg. liminatans reacted only with the homologous enzyme and did not form precipitates with NADP(+)-dependent ADHs. No immunological relation of the NADP(+)- or F420-dependent ADHs of methanogens with ADH of yeast or horse liver was found. In accordance with the immunological data, the N-terminal amino acid sequences of the NADP(+)-dependent ADHs of Mb. palustre and Mcp. parvum had a high degree of similarity, whereas the N-terminal amino acid sequence of the ADH of Mg. liminatans revealed no similarity with the two NADP(+)-dependent enzymes.

Alcohol Oxidoreductases↗
PubMed1991

Interaction of ferredoxin-NADP+ reductase from Anabaena with its substrates.

The interaction of ferredoxin-NADP+ reductase from the cyanobacterium Anabaena variabilis with its substrates, NADP+ and ferredoxin, has been studied by difference absorption spectroscopy. Several structural analogs of NADP+ have been shown to form complexes the stabilities of which are strongly dependent on the ionic strength of the medium. In most cases the binding energy of these complexes and their difference absorption spectra are similar to those reported for the spinach enzyme. However, NADP+ perturbs the absorption spectra of the Anabaena and spinach enzymes in a different way. This difference has been shown to be related to the binding of the nicotinamide ring of NADP+ to the enzymes. These results are interpreted as being due to a different nicotinamide binding site in the two reductases. The enthalpic and entropic components of the Gibbs energy of formation of the NADP+ complex have been estimated. An increase in entropy on NADP+ binding seems to be the main source of stability for the complex. A shift of approximately 40 mV in the redox potential of the couple NADP+/NADPH has been observed to occur upon binding of NADP+ to the oxidized enzyme. This allows us to calculate the binding energy between the reductase and NADPH. The ability of the reductase, ferredoxin, and NADP+ to form a ternary complex indicates that the protein carrier binds to the reductase through a different site than that of the pyridine nucleotide.

Binding Sites↗
PubMed2003

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme.

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.

Alkaline Phosphatase↗
PubMed1997

Caged NADP and NAD. Synthesis and characterization of functionally distinct caged compounds.

Two caged NADP compounds have been synthesized and characterized for use in the crystallographic study of isocitrate dehydrogenase (IDH), as well as for general use in cell biology, metabolism, and enzymology. One caged NADP compound has been designed to be "catalytically caged" so that it can bind to IDH prior to photolysis but is not catalytically active. A second NADP compound is "affinity caged" so that addition of the caging group inhibits binding of the compound to IDH prior to photolysis. The catalytically caged compound was synthesized in a two-step process, starting with the NADase-catalyzed exchange of a synthetic nicotinamide derivative onto NADP. X-ray structures of the NADP compounds with IDH show the catalytically caged NADP bound to the enzyme with its nicotinamide group improperly positioned to allow turnover, while the affinity caged NADP does not bind to the enzyme at concentrations up to 50 mM. Two analogous caged NAD compounds have also been synthesized. The NADP and NAD compounds were characterized in terms of kinetics, quantum yield, and product formation. The affinity caged NADP compound P2'-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl] NADP (VIII) is photolyzed at a rate of 1.8 x 10(4) s-1 with a quantum yield of 0.19 at pH 7; the NAD analog P-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl] NAD (IX) is photolyzed at at a rate of 1.7 x 10(4) s-1 with a quantum yield of 0.17.

Binding Sites↗
PubMed2000

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues.

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.

Animals↗
PubMed2002

NADP-malate dehydrogenase from Chlamydomonas: prediction of new structural determinants for redox regulation by homology modelling.

The function of a gene closely linked to nitrate assimilation loci from Chlamydomonas reinhardtii has been investigated. Gene expression analysis shows that its mRNA accumulation is modulated by light, carbon source and adaptation to light/dark cyclic conditions of growth. A full-length cDNA was isolated for the light-regulated transcript, and sequence characterization indicates that it encodes the NADP-malate dehydrogenase from C. reinhardtii (NADP-MDH;Cr). The primary structure of NADP-MDH;Cr is closely related to plant, mossfern and algal NADP-malate dehydrogenases, and shares structural determinants for chloroplast targeting, cofactor binding and catalysis. Sequence conservation extends to the carboxy end of the protein, where plant and mossfern enzymes have two cysteines and an acidic C-terminus with a critical role for regulation of NADP-MDH activity by the thioredoxin/ferredoxin system. Accordingly, incubation with DTT activates NADP-MDH enzyme in cell-free extracts from C. reinhardtii. Like NADP-malate dehydrogenases from two other green algae, the N-terminal extension of NADP-MDH;Cr lacks two thiol residues whose reduction constitutes the rate-limiting step in the activation reaction of plant enzymes. Homology-based 3D modelling of NADP-MDH;Cr, the first structure predicted for NADP-malate dehydrogenase from a lower eukaryote, evidences close positioning of two new cysteines in an accessible region of the protein surface. These results suggest that the algal enzyme has a different arrangement of regulatory disulfide bridges, which might involve the existence of new mechanisms that control functioning of the malate valve, the main system to export reducing power from the chloroplast of plant cells.

Algal Proteins↗