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PubMed2003

Inhibition of the group I ribozyme splicing by NADP+.

The coenzyme NADP+ (nicotinamide adenine dinucleotide phosphate) and its analogs were tested for inhibition of the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td). Of all compounds examined, the 3'-NADP was the most potent inhibitor and the order of inhibitory efficiency for compounds tested was as follows: 3'-NADP+ > NADP+ > NADP(+)-dialdehyde > NADPH > 1,N6-etheno-NADP+. Increasing guanosine concentration up to 40 microM overcame the suppression of self-splicing by NADP+ up to 76% of the level of normal splicing but didn't recover the full splicing activity. Similarly, Mg2+ also served to restore the splicing activity by about 90% at 25 mM concentration above which the splicing started to decline. The kinetic analysis showed that NADP+ acts as a mixed type non-competitive inhibitor for the td intron RNA with a Ki of 4.1 mM. The specificity of the splicing inhibition by NADP+ is predominantly due to increases in Km and decreases in kcat values. The results indicate that the inhibition by NADP+ was guanosine and Mg2+ dependent.

Kinetics↗
PubMed2005

Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenase.

Human glucose-6-phosphate dehydrogenase (G6PD) is NADP(+)-dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, DeltaG6PD, with glucose-6-phosphate and NADP(+) have been crystallized and their structures solved to 2.9 and 2.5 A, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PD(Canton)) in which NADP(+) is bound at the structural site. Substrate binding in DeltaG6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP(+) binding at the coenzyme site is seen to be comparable to NADP(+) binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer-dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP(+) binds in a very similar way in the DeltaG6PD-NADP(+) complex and in G6PD(Canton), while in the substrate complex the structural NADP(+) has low occupancy and the C-terminal tail at the structural NADP(+) site is disordered. The implications of possible interaction between the structural NADP(+) and G6P are considered.

Binding Sites↗
PubMed1985

Actions of adenine dinucleotides in the guinea-pig taenia coli: NAD acts indirectly on P1-purinoceptors; NADP acts like a P2-purinoceptor agonist.

The actions of the adenine dinucleotides beta-nicotinamide adenine dinucleotide (NAD) and beta-nicotinamide adenine dinucleotide phosphate (NADP) were examined on the carbachol-contracted taenia coli of the guinea-pig. Both were capable of inducing full relaxations in a concentration-dependent manner; NADP was 21.4 times more effective than NAD at EC50; the threshold for NADP was approximately 0.1 microM and for NAD approximately 1.0 microM. The P1-purinoceptor antagonist, 8-phenyltheophylline (10 microM), produced a large parallel rightward shift in the NAD concentration-response curve; in contrast it produced a small parallel leftward shift in the NADP concentration-response curve. Dipyridamole (0.2 microM), a purine nucleoside uptake inhbitor, markedly potentiated responses to NAD and slightly potentiated NADP. 8-Phenyltheophylline antagonized the dipyridamole potentiation of both NAD and NADP. By use of high performance liquid chromatography it was shown that the action of NAD involves a breakdown to adenosine. Apamin, a K+ channel blocker, which antagonizes P2-purinoceptor activation in the intestine, abolished responses to NADP but not to NAD. The alpha-and beta-adrenoceptor antagonists, phentolamine (1 microM) and propranolol (1 microM), did not affect responses to NAD or NADP. Tetrodotoxin, a neurotoxin, did not abolish responses to either NAD or NADP. It is concluded that NAD acts largely indirectly as a P1-purinoceptor agonist following its breakdown to adenosine by ectoenzymes, while NADP acts in a similar manner to a P2-purinoceptor agonist.

Adenosine↗
PubMed1998

Purification and characterization of NADP+-linked isocitrate dehydrogenase from scots pine . Evidence for different physiological roles of the enzyme in primary development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (10(5) M-1 s-1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.

Journal Article↗
PubMed1996

Properties of the purified, recombinant, NADP(H)-binding domain III of the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum.

Transhydrogenase comprises three domains. Domains I and III are peripheral to the membrane and possess the NAD(H)- and NADP(H)-binding sites, respectively, and domain II spans the membrane. Domain III of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified. The purified protein was associated with substoichiometric quantities of tightly bound NADP+ and NADPH. Fluorescence spectra of the domain III protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close. The rate constants for NADP+ release and NADPH release from domain III were 0.03 s-1 and 5.6 x 10(4) s-1, respectively. In the absence of domain II a mixture of the recombinant domain III protein, plus the previously described recombinant domain I protein, catalysed reduction of acetylpyridine-adenine dinucleotide (AcPdAD+) by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP+ from domain III. Similarly, the mixture catalysed reduction of thio-NADP+ by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain III. The mixture also catalysed very rapid reduction of AcPdAD+ by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain III and vice versa indicated (a) that during reduction of AcPdAD+ by NADPH, a single domain I protein can visit and transfer H equivalents to about 60 domain III proteins during the time taken for a single domain III to release its NADP+, whereas (b) the cyclic reaction is rapid on the timescale of formation and break-down of the domain I. III complex. The rate of the hydride transfer reaction was similar in the domain I.III complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I.III complex due to the greatly decreased rates of release of NADP+ and NADPH. It is concluded that, in the complete enzyme, conformational changes in the membrane-spanning domain II, which result from proton translocation, lead to changes in the binding affinity of domain III for NADP+ and for NADPH.

Binding Sites↗
PubMed2006

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase.

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

Bacterial Proteins↗
PubMed2001

Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase.

ADP-L-glycero-D-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-l-glycero-D-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD(+) cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000) Structure 5, 453-462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD(+), NADP(+), and FAD. In this report we provide data that shows NAD(+) and NADP(+) both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP(+) over NAD(+). The K(d) value for NADP(+) was 26 microm whereas that for NAD(+) was 45 microm. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP(+) had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD(+). Perchloric acid extracts of the purified enzyme were assayed with NAD(+)-specific alcohol dehydrogenase and NADP(+)-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP(+) in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD(+) cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP(+).

Adenine Nucleotides↗
PubMed2003

Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+: insight into the cofactor recognition, catalysis, and evolution.

NADP+-dependent monomeric isocitrate dehydrogenase (IDH) from the nitrogen-fixing bacterium Azotobacter vinelandii (AvIDH) is one of members of the beta-decarboxylating dehydrogenase family and catalyzes the dehydration and decarboxylation of isocitrate to yield 2-oxoglutrate and CO2 in the Krebs cycle. We solved the crystal structure of the AvIDH in complex with cofactor NADP+ (AvIDH-NADP+ complex). The final refined model shows the closed form that has never been detected in any previously solved structures of beta-decarboxylating dehydrogenases. The structure also reveals all of the residues that interact with NADP+. The structure-based sequence alignment reveals that these residues were not conserved in any other dimeric NADP+-dependent IDHs. Therefore the NADP+ specificity of the monomeric and dimeric IDHs was independently acquired through the evolutional process. The AvIDH was known to show an exceptionally high turnover rate. The structure of the AvIDH-NADP+ complex indicates that one loop, which is not present in the Escherichia coli IDHs, reliably stabilizes the conformation of the nicotinamide mononucleotide of the bound NADP+ by forming a few hydrogen bonds, and such interactions are considered to be important for the monomeric enzyme to initiate the hydride transfer reaction immediately. Finally, the structure of the AvIDH is compared with that of other dimeric NADP-IDHs. Several structural features demonstrate that the monomeric IDHs are structurally more related to the eukaryotic dimeric IDHs than to the bacterial dimeric IDHs.

Amino Acid Sequence↗
PubMed1988

Mutations affecting the synthesis of NADP-dependent glutamate dehydrogenase in Pseudomonas aeruginosa.

NADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The Mr determined by Sephadex gel filtration was 280,000; the subunit Mr determined by SDS-PAGE was 45,000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh-Glt-) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).

Genetic Linkage↗
PubMed1990

NADP+ reduction by human lymphocytes.

The hexose monophosphate shunt (HMPS) is known to be responsible for the reduction of NADP+ by lymphocytes. We tried to find other enzymatic systems that might provide the lymphocytes with NADPH. By measuring the absorbance at 340 nm we noted that the addition of NADP+ to a preparation of disrupted lymphocytes resulted in the formation of NADPH at a rate of 4 nmol/10(6) cells per min. This phenomenon could not be changed by negative feedback inhibition of HMPS, and could not be attributed to the low concentration of glucose, glucose-6-phosphate (G-6-P) and isocitrate found in the cell preparation (NADP(+)-dependent isocitrate dehydrogenase in addition to HMPS NADP+ reducing enzymes was found to be present in lymphocytes). Because of the activity of a NADP(+)-dependent lactate dehydrogenase, pyruvate oxidized the NADPH as it was being formed. Here we demonstrate the presence of an unknown NADP+ reducer in lymphocytes which seems to play an additional role to HMPS in NADP+ reduction by lymphocytes. NADP(+)-dependent lactate dehydrogenase may play a role in regulating the NADP+/NADPH ratio.

Humans↗
PubMed1994

Expression, assembly and secretion of a fully active plant ferredoxin-NADP+ reductase by Saccharomyces cerevisiae.

The flavoprotein ferredoxin-NADP+ reductase catalyzes the final step of the photosynthetic electron transport i.e., the reduction of NADP+ by ferredoxin. Expression and secretion of this enzyme was examined in Saccharomyces cerevisiae using a cDNA cloned from a pea library [Newman, B. J. & Gray, J. C. (1988) Plant Mol. Biol. 10, 511-520]. Two pea library cDNA sequences were employed, one corresponding to the mature enzyme and the other containing, in addition, the sequence of the transit peptide that directs ferredoxin-NADP+ reductase to the chloroplast. These sequences were introduced into a yeast shuttle vector in frame with the mating factor alpha 1 secretion-signal coding region under the control of its natural mating factor alpha 1 promoter. Saccharomyces cerevisiae cells transformed with the recombinant plasmids were able to synthesize and secrete fully active pea ferredoxin-NADP+ reductase. In both cases, a 35-kDa polypeptide was the major product. N-terminal sequencing of the secreted proteins indicates processing at position -1 with respect to the N-terminus of the pea mature enzyme. Yeast cells transformed with plasmid encoding the ferredoxin-NADP+ reductase precursor secrete four-times more ferredoxin-NADP+ reductase to the medium than cells transformed with the plasmid encoding the mature form of the enzyme. Ferredoxin-NADP+ reductases purified from culture medium showed structural and enzymatic properties that were identical, within the experimental error, to those of native plant ferredoxin-NADP+ reductase. The overall results indicate that pea ferredoxin-NADP+ reductase can be properly folded and its prosthetic group assembled in the yeast endoplasmic reticulum, and that its natural transit peptide favors its secretion.

Amino Acid Sequence↗
PubMed1995

Horse liver alcohol dehydrogenase can accept NADP+ as coenzyme in high concentrations of acetonitrile.

The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCl pH 7.4, was investigated. The enzyme could accept NADP+ as coenzyme if it was first bio-imprinted with NADP+, i.e. precipitated from an aqueous buffer with 1-propanol in the presence of NADP+ dried, and then assayed in a system with less than 10% buffer. When assayed in a system with 25% buffer, no activity with NADP+ as coenzyme was observed. The activity was measured with a coenzyme regeneration assay with cinnamoyl alcohol and octylaldehyde as substrates. Other methods to prepare a binary complex of horse liver alcohol dehydrogenase and NADP+, i.e. if the enzyme was immobilized on silica and NADP+ added afterwards or if it was deposited on Celite together with NADP+, failed to show any bio-imprinting effect. The activity of horse liver alcohol dehydrogenase bioimprinted with NADP+ and assayed in acetonitrile/buffer mixtures with less than 10% buffer showed the same or higher activity than an enzyme preparation prepared by bio-imprinting with NAD+. The hypothesis is that the conformation of the active site of horse liver alcohol dehydrogenase is modified to one that is complementary to the ligand present during precipitation and drying. This is possible because of the restricted motility of the enzyme conformation in high concentrations of organic solvent. In the presence of more than 10% buffer the mobility increases in such a way that the imposed conformation with activity towards NADP+ disappears.

Acetonitriles↗
PubMed1990

Photochemical and enzymatic redox transformations of reduced forms of coenzyme NADP+.

The two reduced forms of NADP+, NADPH and its dimer (NADP)2, on irradiation in aqueous medium at 365 nm, are converted to enzymatically active NADP+, with accompanying formation of H2O2. The rate photooxidation of NADPH is strongly dependent on the presence of oxygen, but that of (NADP)2 is similar under aerobic and anaerobic conditions. In the presence of oxygen, but not in its absence, O2-. is an intermediate in the reaction. Generation of H2O2 under anaerobic conditions, confirmed by the fact that presence of peroxidase in irradiated solutions of (NADP)2 enhances photooxidation of the latter, is ascribed to attack on water of the excited dimer. Under anaerobic conditions at pH 9.5, Fe(EDTA)2+ and Fe(CN)4-(6) increase the rate of photooxidation of NADP dimer two-fold. gamma-Irradiation of (NADP)2 at pH 9.5 in the presence of N2O results in 80% conversion to enzymatically active NADP+. A mechanism for photooxidation of (NADP)2 under anaerobic conditions is suggested, and some relevant biological implications are presented.

Catalase↗
PubMed1981

Correlated biochemical and cytochemical studies of nicotinamide adenine dinucleotide phosphatase (NADPase) activity in ameloblasts using structural analogues of NADP.

The effects of altering the molecular structure of nicotinamide adenine dinucleotide phosphate (NADP) on enzymatic hydrolysis of the monoester phosphate group was examined at pH 5.0 in rat incisor ameloblasts using eight analogues of the oxidized form of the beta-isomer of NADP (beta-NADP+) modified in either the nicotinamide or adenine regions, or at the site for attachment of the monoester phosphate group to the molecule. Biochemical studies with whole homogenates of unfixed enamel organs revealed that the Michaelis-Menten constant (KM) and the maximum rate of dephosphorylation (Vmax) were different for these analogues relative to values estimated with beta-NADP+ as substrate. For example, the KM value was 3-fold higher and the Vmax value was lower by about 1/2 with the reduced form of the molecule as substrate. The KM value was about 2-fold higher but the Vmax value was about the same with an analogue lacking the nicotinamide group as substrate, while both the KM and the Vmax values were about 2-fold higher with an analogue containing an ethylene group covalently linked to adenine as substrate. In contrast, the KM value was markedly reduced (1/15) and the Vmax value was elevated (4-fold) using an analogue as substrate which contained the phosphate group attached at the 3'-position (3'-NADP+), rather than the 2'-position as in beta-NADP+ and other analogues. Cytochemical studies with glutaraldehyde-fixed enamel organs revealed that reaction product from hydrolysis of beta-NADP+ and other analogues containing the phosphate group attached at the natural 2'-position was localized within the intermediate saccules of the ameloblast Golgi apparatus. Reaction product from hydrolysis of 3'-NADP+, however, was localized at a different site, that is, within granules and membranous connections of the GERL system in the cell. Hence, 3'-NADP+ was not hydrolyzed by NADPase but by another enzyme tentatively identified as Coenzyme A phosphatase.

Ameloblasts↗
PubMed1991

A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana.

Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit.

Amino Acid Sequence↗
PubMed1995

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast, Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase.

Peroxisomal NADP-linked isocitrate dehydrogenase (Ps-NADP-IDH) was purified for the first time from Candida tropicalis cells grown on n-alkane as a carbon source, which was effective in proliferation of peroxisomes. The properties of Ps-NADP-IDH were compared with those of mitochondrial NAD-linked isocitrate dehydrogenase (Mt-NAD-IDH) purified from the cells grown on acetate, in which peroxisomes did not proliferate. Ps-NADP-IDH was a homodimer of identical subunits (45 kDa), while Mt-NAD-IDH was suggested to be a heterooctamer composed of two types of subunits with different molecular masses (41 and 38 kDa). Kinetic studies revealed that Ps-NADP-IDH gave Michaelis-Menten saturation curves against isocitrate and NADP concentrations, whereas Mt-NAD-IDH was an allosteric enzyme regulated by ATP, AMP, and citrate. Inhibition by 2-oxoglutarate, a precursor of glutamate, was observed only for Ps-NADP-IDH. Both enzymes were inhibited by concomitant addition of oxalacetate and glyoxylate. The function of Ps-NADP-IDH seems to be completely discriminated from that of Mt-NAD-IDH as reflected by their distinct subcellular localizations. Furthermore, the properties of Ps-NADP-IDH were also compared with those of other mitochondrial and cytosolic IDHs from sources reported previously.

Acetates↗
PubMed2005

MJ0917 in archaeon Methanococcus jannaschii is a novel NADP phosphatase/NAD kinase.

NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.

Archaeal Proteins↗
PubMed2003

Light activation of NADP malic enzyme in leaves of maize: marginal increase in activity, but marked change in regulatory properties of enzyme.

This article reports the characteristics of light activation of NADP-malic enzyme (NADP-ME, EC 1.1.1.40) in leaf discs of maize (Zea mays cv. VMH 404) for the first time. The leaf discs were illuminated in the presence of 2 mmol/L bicarbonate, as light activation increases in the presence of bicarbonate. Upon illumination, the Vmax of NADP-ME increased by about 30%. Although small, the increase was consistent and significant. The changes in regulatory properties of NADP-ME were quite pronounced. The extent of light activation was similar when substrate (malate) concentration was either 4 mmol/L (saturating) or 0.01 mmol/L (limiting). There was only a marginal change in the Km for malate, but there was marked change in the response of NADP-ME to activators or inhibitors. The Ki for pyruvate and oxalate increased by 100 and 67% respectively, while the Ka for the citrate and succinate increased by 36 and 32% respectively. These results suggest that the NADP-ME becomes less sensitive to feedback inhibition on illumination. The light-induced change seems to be due, at least partially, to the reduction of dithiols, as incubation of leaf extracts with DTE dampened light activation of NADP-ME. We conclude that the properties of NADP-ME do change on illumination. Although there was only a marginal increase in the activity of the enzyme on illumination of leaf discs, the changes in regulatory properties of NADP-ME were marked.

Enzyme Activation↗