The simultaneous determination of NAD(H) and NADP(H) utilization by glutamate dehydrogenase

Glutamate dehydrogenase (GDH) from vertebrates is unusual among NAD(P)H-dependent dehydrogenases in that it can use either NAD(H) or NADP(H) as cofactor. In this study, we measure the rate of cofactor utilization by bovine GDH when both cofactors are present. Methods for both reaction directions were developed, and for the first time, to our knowledge, the GDH activity has been simultaneously studied in the presence of both NAD(H) and NADP(H). Our data indicate that NADP(H) has inhibitory effects on the rate of NAD(H) utilization by GDH, a characteristic of GDH not previously recognized. The response of GDH to allosteric activators in the presence of NAD(H) and NADP(H) suggests that ADP and leucine moderate much of the inhibitory effect of NADP(H) on the utilization of NAD(H). These results illustrate that simple assumptions of cofactor preference by mammalian GDH are incomplete without an appreciation of allosteric effects when both cofactors are simultaneously present.     

Rhythmic changes in the activities of NAD kinase and NADP phosphatase in the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z)

NAD kinase and NADP phosphatase activities were detected in the supernatant and the pellet fractions prepared by sonication and centrifugation of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis. A detailed study of substrate concentration-velocity curves enabled us to define the saturating substrate concentrations that were used in the enzyme assays. An analysis of the reproducibility of the entire assay procedure indicated that the pooled standard error was about 14%. We report circadian variations in the activities of NAD kinase and NADP phosphatase in the soluble and membrane-bound fractions of both synchronously dividing and nondividing cultures maintained in constant darkness. Bimodal circadian rhythms in total NADP phosphatase activity were found in dividing cells (peaks at circadian times [CT] 00 and 12). The peak observed at CT 00-03 disappeared when the cells had ceased dividing, a result that suggests that it might be regulated by the cell division cycle. NAD kinase activity displayed unimodal circadian rhythms (peak at CT 12) in dividing cells, which persisted with the same phase after the culture entered the stationary phase of growth. Results are discussed with reference to a model (K. Goto, D. L. Laval-Martin, and L. N. Edmunds, Jr., 1985, Science 228, 1284-1288) in which we have proposed that the Ca2(+)-transport system, Ca2+, calmodulin, NAD kinase, and NADP phosphatase could represent clock "gears" that might constitute a self-sustained circadian oscillating loop.     

NADP(H) phosphatase activities of archaeal inositol monophosphatase and eubacterial 3'-phosphoadenosine 5'-phosphate phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP+ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3'-phosphoadenosine 5'-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3'-phosphoadenosine 5'-phosphate phosphatase. Based on this fact, we found that 3'-phosphoadenosine 5'-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3'-phosphoadenosine 5'-phosphate phosphatase rather than as NADP(H) phosphatase.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis

Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E. coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping. Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet. The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine [Prasad, G. S., et al. (1999) Nat. Struct. Biol. 6, 1126-1131] and human heart [White, A. W., et al. (2000) Structure 8, 1-12] transhydrogenases. Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates. To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI). All mutants except R425C showed altered NADP(H) binding and domain interaction properties. In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI. Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context.     

NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K _m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn²⁺ and Ca²⁺ could be substituted for Mg²⁺. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K _i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist     

Different functions assigned to NAD(H) and NADP(H) in light-dependent nitrogen fixation by heterocysts of Anabaena variabilis

Abstract We have directly selected thermosensitive alleles of cya and crp genes from the wild-type, and present preliminary characterization of these mutants. The selection procedure is based on prior growth of a mutagenized wild-type culture in a medium that counterselects, at low temperature, non-conditional relative to thermosensitive mutants, followed by routine selection of mutants at high temperature. This method should be applicable to various genetic systems.     

即时浸酸显著提高滞育性家蚕卵辅酶Ⅰ和Ⅱ含量

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 提高了其呼吸耗氧量, 抑制了山梨醇积累。本研究利用HPLC法测定了家蚕滞育卵和5 min即时浸酸滞育性卵中辅酶Ⅰ和Ⅱ含量。结果表明： 产下后24-72 h, 家蚕滞育卵中NAD, NADH, NADP和NADPH含量分别下降了30%, 37%, 50%和4%; 而即时浸酸滞育性卵中分别增加了77%, 46%, 142%和241%。不过, 即时浸酸并未显著改变滞育性家蚕卵中NADH/NAD和NADPH/NADP比值。据此推测, 即时浸酸提高滞育性家蚕卵辅酶Ⅰ含量与其呼吸耗氧量增加有关; 即时浸酸显著提高辅酶Ⅱ含量与山梨醇积累抑制无关, 而主要与生物合成加强有关。     

Flow-cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels

A cytofluorimeter is described, using the combination of Argon UV (351-363 nm) and Argon Blue (488 nm) lasers. The dual excitation makes it possible to monitor simultaneously the redox state of flavins and NAD(P)H as indicative of cell metabolic state. Light scatter, absorption, and staining with exogenous fluorescent dyes can add additional information. Thus, a five-parameter flow analysis becomes possible. The present paper describes flavin and NAD(P)H measurements on isolated rat liver cells and mouse bone marrow.     

Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD+ leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP+. Accordingly, the enzyme with the Asp(69)-->Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68)-->Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP+. The Glu(68)-->Lys:Asp(69)-->Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.     

Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex

Eleven genes (ndhA-ndhK) encoding proteins homologous to the subunits of bacterial and mitochondrial NADH dehydrogenase (complex I) were found in the plastid genome of most land plants. These genes encode subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in photosystem I (PSI) cyclic electron transport and chlororespiration. Although the chloroplast NDH is believed to be closely and functionally related to the cyanobacterial NDH-1L complex, extensive proteomic, genetic and bioinformatic studies have discovered many novel subunits that are specific to higher plants. On the basis of extensive mutant characterization, the chloroplast NDH complex is divided into four parts, the A, B, membrane and lumen subcomplexes, of which subunits in the B and lumen subcomplexes are specific to higher plants. These results suggest that the structure of NDH has been drastically altered during the evolution of land plants. Furthermore, chloroplast NDH interacts with multiple copies of PSI to form the unique NDH-PSI supercomplex. Two minor light-harvesting-complex I (LHCI) proteins, Lhca5 and Lhca6, are required for the specific interaction between NDH and PSI. The evolution of chloroplast NDH in land plants may be required for development of the function of NDH to alleviate oxidative stress in chloroplasts. In this review, we summarize recent progress on the subunit composition and structure of the chloroplast NDH complex, as well as the information on some factors involved in its assembly. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Mathematical modeling of in vitro enzymatic production of 2-Keto-L-gulonic acid using NAD(H) or NADP(H) as cofactors

A 2-Keto-L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis. Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor. An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed. A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor. The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost. The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor. The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations. This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.     

Structure and function of regulatory RNA elements: Ribozymes that regulate gene expression

Since their discovery in the 1980s, it has gradually become apparent that there are several functional classes of naturally occurring ribozymes. These include ribozymes that mediate RNA splicing (the Group I and Group II introns, and possibly the RNA components of the spliceosome), RNA processing ribozymes (RNase P, which cleaves precursor tRNAs and other structural RNA precursors), the peptidyl transferase center of the ribosome, and small, self-cleaving genomic ribozymes (including the hammerhead, hairpin, HDV and VS ribozymes). The most recently discovered functional class of ribozymes include those that are embedded in the untranslated regions of mature mRNAs that regulate the gene's translational expression. These include the prokaryotic glmS ribozyme, a bacterial riboswitch, and a variant of the hammerhead ribozyme, which has been found embedded in mammalian mRNAs. With the discovery of a mammalian riboswitch ribozyme, the question of how an embedded hammerhead ribozyme's switching mechanism works becomes a compelling question. Recent structural results suggest several possibilities.     

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.     

Tetrahydrobiopterin biosynthesis. Studies with specifically labeled (2H)NAD(P)H and 2H2O and of the enzymes involved

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)-(4-2H)NAD(P)H or (S)-(4-2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro-R hydrogen of NAD(P)H during the reduction of 7,8-dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of NADPH during the reduction of sepiapterin to 7,8-dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4-pro-S hydrogen from NADPH is incorporated at each of the C(1') and C(2') position of BH4. Label from the solvent is also introduced into position C(3'). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6-pyruvoyl-tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihyroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6-pyruvoyl-tetrahydropterin might be catalyzed by sepiapterin reductase.     

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

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

Extremely high intracellular concentration of glucose-6-phosphate and NAD(H) in Deinococcus radiodurans

Extremophiles - Deinococcus radiodurans is highly resistant to ionizing radiation and UV radiation, and oxidative stress caused by such radiations. NADP(H) seems to be important for this resistance...