Regeneration Of NADP Research Articles

Two-step d-ononitol epimerization pathway in Medicago truncatula.

Methylated inositol, d-pinitol (3-O-methyl-d-chiro-inositol), is a common constituent in legumes. It is synthesized from myo-inositol in two reactions: the first reaction, catalyzed by myo-inositol-O-methyltransferase (IMT), consists of a transfer of a methyl group from S-adenosylmethionine to myo-inositol with the formation of d-ononitol, while the second reaction, catalyzed by d-ononitol epimerase (OEP), involves epimerization of d-ononitol to d-pinitol. To identify the genes involved in d-pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co-expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo-keto reductase and MtOEPB, encoding a short-chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E.coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo-inositol to d-ononitol and showed that MtOEPA enzyme has NAD+ -dependent d-ononitol dehydrogenase activity, while MtOEPB enzyme has NADP+ -dependent d-pinitol dehydrogenase activity. Both enzymes are required for epimerization of d-ononitol to d-pinitol, which occurs in the presence of NAD+ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d-ononitol and d-pinitol in transformants. As this two-step pathway of d-ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD+ , we speculate that one of the functions of this pathway might be regeneration of NADP+ during drought stress.

The metabolic changes in tumor-associated macrophages during cancer grow in mice with Ehrlich ascites carcinoma.

In this work, we investigated the activity of the key NAD(P)-dependent dehydrogenases associated with macrophage tumors in mice with Ehrlich ascites carcinoma. It was shown that cancer grow is associated with the development of conditions in macrophages leading to a decrease in the substrate flow intensity in the tricarboxylic acid cycle, deceleration of oxidative deamination of L-glutamate, NADP regeneration, and a decrease in the antioxidant defense efficiency. There results are consistent with our recent concept on the nonspecific metabolic reaction of cells to extreme exposures.

Cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids.

A gene encoding a novel 7α-specific NADP+-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP+ using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.

Effect of Tween 40 and DtsR1 on L-arginine overproduction in Corynebacterium crenatum.

Backgroundl-Glutamate is an important precursor in the l-arginine (l-Arg) biosynthetic pathway. Various methods, including polyoxyethylene sorbitan monopalmitate (Tween 40) addition and dtsR1 disruption, have been widely used to induce l-glutamate overproduction in Corynebacterium glutamicum. In this study, a novel strategy for l-Arg overproduction through Tween 40 trigger and ΔdtsR1 mutant were proposed in Corynebacterium crenatum.ResultsCorynebacterium crenatum mutant (CCM01) was selected as a host strain, whose argR was lethal via mutagenesis screening, the proB gene was knocked out, and argB was replaced by argB M4 (E19R, H26E, D311R, and D312R) to release l-Arg feedback resistance. After Tween 40 trigger in the logarithmic period, l-Arg production increased from 15.22 to 17.73 g/L in CCM01 strain. When NCgl1221 and dtsR1 disruption (CCM03), l-Arg production drastically increased to 27.45 g/L and then further to 29.97 g/L after Tween 40 trigger. Moreover, the specific activity of α-oxoglutarate dehydrogenase complex (ODHC) decreased, whereas the regeneration of NADP+/NADPH significantly increased after dtsR1 disruption and Tween 40 trigger. Results of real-time PCR showed that the transcriptional levels of odhA, sucB, and lpdA (encoding three subunits of the ODHC complex) were downregulated after Tween 40 trigger or dtsR1 disruption. By contrast, zwf transcription (encoding glucose-6-phosphate dehydrogenase) showed no significant difference among CCM01, CCM02 (ΔNCgl1221), and CCM03 (ΔNCgl1221ΔdtsR1) strains without Tween 40 trigger but evidently increased by 5.50 folds after Tween 40 trigger.ConclusionA novel strategy for l-Arg overproduction by dtsR1 disruption and Tween 40 trigger in C. crenatum was reported. Tween 40 addition exhibited a bifunctional mechanism for l-Arg overproduction, including reduced ODHC activity and enhanced NADPH pools accumulation by downregulated dtsR1 expression and upregulated zwf expression, respectively.

Gene Regulatory and Metabolic Adaptation Processes of Dinoroseobacter shibae DFL12T during Oxygen Depletion

Metabolic flexibility is the key to the ecological success of the marine Roseobacter clade bacteria. We investigated the metabolic adaptation and the underlying changes in gene expression of Dinoroseobacter shibae DFL12(T) to anoxic life by a combination of metabolome, proteome, and transcriptome analyses. Time-resolved studies during continuous oxygen depletion were performed in a chemostat using nitrate as the terminal electron acceptor. Formation of the denitrification machinery was found enhanced on the transcriptional and proteome level, indicating that D. shibae DFL12(T) established nitrate respiration to compensate for the depletion of the electron acceptor oxygen. In parallel, arginine fermentation was induced. During the transition state, growth and ATP concentration were found to be reduced, as reflected by a decrease of A578 values and viable cell counts. In parallel, the central metabolism, including gluconeogenesis, protein biosynthesis, and purine/pyrimidine synthesis was found transiently reduced in agreement with the decreased demand for cellular building blocks. Surprisingly, an accumulation of poly-3-hydroxybutanoate was observed during prolonged incubation under anoxic conditions. One possible explanation is the storage of accumulated metabolites and the regeneration of NADP(+) from NADPH during poly-3-hydroxybutanoate synthesis (NADPH sink). Although D. shibae DFL12(T) was cultivated in the dark, biosynthesis of bacteriochlorophyll was increased, possibly to prepare for additional energy generation via aerobic anoxygenic photophosphorylation. Overall, oxygen depletion led to a metabolic crisis with partly blocked pathways and the accumulation of metabolites. In response, major energy-consuming processes were reduced until the alternative respiratory denitrification machinery was operative.

ChemInform Abstract: Synthesis of Optically Active α‐Bromohydrins via Reduction of α‐Bromoacetophenone Analogues Catalyzed by an Isolated Carbonyl Reductase.

AbstractA new selective synthesis of (S)‐α‐bromohydrins is presented, involving the highly stereoselective (&lt;99% e.e.) reduction of α‐bromoacetophenones (I) with carbonyl reductase in a D‐glucose—D‐glucose dehydrogenase‐based NADP regeneration system.

A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor

AbstractThe NAD+‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD+ and exhibits only a low activity with NADP+ as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD+. While an application in the regeneration of NADP+ is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP+‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP+ acceptance. The mutant V39R was identified to have increased activity with NADP+ relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP+ in an enzyme‐coupled approach combined with the NADP+‐dependent alcohol dehydrogenase from Lactobacillus kefir. We demonstrate for the first time an application of an LDH in the regeneration of NADP+.

Electrochemical regeneration of NADP+ using metal oxide electrodes and its application for organic synthesis

Electrochemical regeneration of NADP+ using metal oxide electrodes and its application for organic synthesis

Regeneration of the nicotinamide cofactor using a mediator-free electrochemical method with a tin oxide electrode

Tin (IV) oxide was made using an anodization and annealing method and was used as a working electrode in an electrochemical cofactor regeneration reaction. This material was formed with a large surface area, and by changing the preparation conditions, it was possible to control the morphology. Tin oxide has redox properties similar to those of frequently used mediators required for electron transfer between cofactors and an electrode. Therefore, by using tin oxide as a novel electrode, mediator-free electrochemical cofactor regeneration may be possible. Oxidation and reduction of the nicotinamide cofactors, NAD(P)H and NAD(P) +, were carried out under various reaction conditions. The results showed a high efficiency for oxidizing NADH over a broad range of pH and temperatures. The oxidation tendency of NADPH was also observed, and it demonstrated a similar reaction tendency as NADH. When using a tin oxide electrode, NAD + was readily reduced to NADH, though the efficiency of this reaction was lower than for NADH oxidation. Oxidation of 2-propanol to acetone was used as a model system using alcohol dehydrogenase and the cofactor regeneration system suggested in this study. The electroenzymatic reaction showed efficient regeneration of NADP + without a mediator.

Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

A soluble ferric reductase, SfrAB, which catalysed the NADPH-dependent reduction of chelated Fe(III), was previously purified from the dissimilatory Fe(III)-reducing micro-organism Geobacter sulfurreducens, suggesting that reduction of chelated forms of Fe(III) might be cytoplasmic. However, metabolically active spheroplast suspensions could not catalyse acetate-dependent Fe(III) citrate reduction, indicating that periplasmic and/or outer-membrane components were required for Fe(III) citrate reduction. Furthermore, phenotypic analysis of an SfrAB knockout mutant suggested that SfrAB was involved in acetate metabolism rather than respiration-linked Fe(III) reduction. The mutant could not grow via the reduction of either Fe(III) citrate or fumarate when acetate was the electron donor but could grow with either acceptor if either hydrogen or formate served as the electron donor. Following prolonged incubation in acetate : fumarate medium in the absence of hydrogen and formate, an 'acetate-adapted' SfrAB-null strain was isolated that was capable of growth on acetate : fumarate medium but not acetate : Fe(III) citrate medium. Comparison of gene expression in this strain with that of the wild-type revealed upregulation of a potential NADPH-dependent ferredoxin oxidoreductase as well as genes involved in energy generation and amino acid uptake, suggesting that NADPH homeostasis and the tricarboxylic acid (TCA) cycle were perturbed in the 'acetate-adapted' SfrAB-null strain. Membrane and soluble fractions prepared from the 'acetate-adapted' strain were depleted of NADPH-dependent Fe(III), viologen and quinone reductase activities. These results indicate that cytoplasmic, respiration-linked reduction of Fe(III) by SfrAB in vivo is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.

Generation and scavenging of reactive oxygen species in chloroplasts: a submolecular approach

The specific traits of oxygen chemistry are examined as a basis of the step-wise electron acceptance by O 2 , giving rise to incompletely reduced highly reactive oxygen species (ROS) comprising both free radical (O 2 _ - ,OH_, HO 2 _) and non-radical (molecular) forms (H 2 O 2 ). The reversal of electron spin of oxygen, induced by energy transfer from triplet state chlorophyll, is considered as a generator of another non-radical ROS, singlet oxygen ( 1 O 2 ). The view that ROS are primarily agents of damage is questioned by data proving their beneficial role and particularly their signaling function. In this context the importance of the equilibrium between ROS production and scavenging, i.e. the regulation of ROS is emphasized. Particular attention is given to the chloroplast, a major source of ROS, which harbors ROS-producing centers (triplet chlorophyll, ETC in PSI and PSII) and a diversified ROS-scavenging network (antioxidants, SOD, APX-glutathione cycle, and a thioredoxin system). The electron carriers for ROS generation are specified. These include Fe-S clusters and ferredoxin in PSI, and Q A and Q B in PSII. The mechanisms and the molecular targets of damaging effect of ROS in chloroplasts are described. The photoprotective role of ROS formation is explained in terms of the use of oxygen as an electron acceptor, as an alternative to NADP, in high-light conditions producing excess electron flow, overreduction of the ETC and overproduction of NADPH. Adequate scavenging of ROS essentially contributes to photoprotection. Stress factors limiting CO 2 fixation and hence decreasing NADPH utilization and NADP regeneration exacerbate high-light stress; ROS formation and scavenging become unbalanced, and oxidative damage may occur. The water-water cycle in chloroplasts and photorespiration as alternative mechanisms of ROS scavenging, NADP regeneration, and dissipation of excess excitation energy are briefly examined. The defensive role of avoidance of ROS generation through alternative oxidases, reducing O 2 directly to water, is also described. The signaling function of ROS is illustrated by data proving the high-light induced systemic induction of APX genes and acclimation phenomena mediated by H 2 O 2 . The use of transgenic approaches for modeling ROS-tolerant plants is discussed.

Photochemical Coenzyme Regeneration in an Enzymatically Active Optical Material

The photoinduced electron transfer between immobilized thionine and the dinucleotide enzyme cofactors NADH and NADPH in a SiO2 sol−gel matrix is reported. The electron-transfer quenching of thionine luminescence is used to monitor the rate of NADPH oxidation. Using Stern−Volmer quenching curves, the quenching rates in the silica matrix are 1 to 2 orders of magnitude smaller than those in solution. The rate constants for oxidation of NADPH by thionine were measured to be 9.8(±2.9) × 10-3 s-1 in solution and 8.8(±1.0) × 10-4 s-1 in the gel. Within the silica matrix, the photoinduced oxidation of NADPH is combined with the enzymatic reaction of isocitrate dehydrogenase, which uses the oxidized cofactor, NADP+, as an electron acceptor in the oxidation of isocitrate. The encapsulated isocitrate dehydrogenase is active with a Michaelis−Menten constant, KM, of 3 μM and a kcat of 0.67 μM/s per mgenzyme. Because optical sensors use NADPH fluorescence as an indicator of the presence and relative concentration of enzyme substrate, the successful demonstration of photoinduced regeneration of NADP+ makes possible continuous monitoring by the family of dehydrogenase enzymes.

Polyphosphate-dependent enzymes in some coryneform bacteria isolated from sewage sludge

Eleven isolates obtained from a laboratory sewage treatment plant, most of them presumptively assigned to the coryneform genera Curtobacterium and Aureobacterium were studied for the presence of intracellular polyphosphates and polyphosphate dependent enzymes. All isolates stored polyphosphates and showed adenylate kinase activities ranging from 64 to 815 mU mg−1. Polyphosphate:AMP phosphotransferase could only be detected in one isolate. Three isolates showed a polyphosphate kinase activity also in minor amounts from 15 to 17 mU mg−1. A polyphosphate dependent NAD or 3-phosphoglycerate kinase could not be detected. Polyphosphate glucokinase activity was measured in cell-free extracts of nine isolates ranging from 2 to 376 mU mg−1. Three isolates showed in addition to the polyphosphate glucokinase, a glucose-6-phosphate-dependent NAD kinase. For the regeneration of NADP from NAD and polyphosphate, this enzyme system may give the isolates a distinct competitive advantage, especially for anabolic processes. The polyphosphate-dependent enzymes reported here may play an additional role in the complex process of ‘biological’ phosphate removal from wastewater.

Polyphosphate-dependent enzymes in some coryneform bacteria isolated from sewage sludge.

Eleven isolates obtained from a laboratory sewage treatment plant, most of them presumptively assigned to the coryneform genera Curtobacterium and Aureobacterium were studied for the presence of intracellular polyphosphates and polyphosphate dependent enzymes. All isolates stored polyphosphates and showed adenylate kinase activities ranging from 64 to 815 mU mg-1. Polyphosphate:AMP phosphotransferase could only be detected in one isolate. Three isolates showed a polyphosphate kinase activity also in minor amounts from 15 to 17 mU mg-1. A polyphosphate dependent NAD or 3-phosphoglycerate kinase could not be detected. Polyphosphate glucokinase activity was measured in cell-free extracts of nine isolates ranging from 2 to 376 mU mg-1. Three isolates showed in addition to the polyphosphate glucokinase, a glucose-6-phosphate-dependent NAD kinase. For the regeneration of NADP from NAD and polyphosphate, this enzyme system may give the isolates a distinct competitive advantage, especially for anabolic processes. The polyphosphate-dependent enzymes reported here may play an additional role in the complex process of 'biological' phosphate removal from wastewater.

Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH

A theoretical model was constructed in order to study charged membrane bioreactors (CMBRs). In this model, it was postulated that a native nicotinamide coenzyme NADP(H) can be partially retained by a charged membrane in continuous operation. A multienzyme system composed of NADPH-dependent aldose reductase (AR) and glucose dehydrogenase (GDH) was used for the production of sorbitol and gluconic acid from glucose and for the conjugated enzymatic regeneration of NADP(H). Both enzymes were studied with respect to their reaction kinetics. AR was determined to obey the Theorell-Chance mechanism. GDH reaction was approximated by the initial velocity equation of the sequential Bi-Bi mechanism since the reverse reaction could be neglected. Significant inhibitions of both enzymes by sorbitol, gluconic acid, and glucose were observed, and the mode of inhibition was estimated to modify the velocity equations. The differential equation system for each component was derived and numerically analyzed according to the model. The theoretical model elucidated several features of the CMBR. (1) When compared at the same productivity, higher retainment was found to bring about a higher coenzyme turnover number, indicating that the feed coenzyme concentration can be reduced. (2) Under constant conversion, a contradictory relationship between turnover number and residence time arises if the feed concentration of a coenzyme varies. The theoretical model predicts that there is a practically optimal concentration for using NADP(H) efficiently. This concentration was consistent with that yielding the estimated minimum total cost. (3) In this system, excess-GDH-to-AR activity was required because of differences in their kinetic constants. The amount of regeneration enzyme required can be reduced by the accumulation of excels NADPH due to coenzyme retainment. (4) Comparison with an ideal repeated batch reaction revealed that the continuously operated CMBR was vastly superior with respect to productivity as well as operation ability.

Lipid Metabolism and Enzyme Activities in Hormone-Dependent and Hormone-Independent Mammary Adenocarcinoma in GR Mice&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

Lipid metabolism in hormone-dependent (HD) GR mouse mammary tumors was compared to that in hormone-independent (HI) tumors and normal mammary tissues. HD tumors, like normal mammary tissue but unlike HI tumors, synthesized medium-chain-length fatty acids (MCFA). However, when treated with hormones (estrone and progesterone), the HI tumors were induced to produce MCFA. The activity of thioesterase II correlated positively with the synthesis of MCFA and was influenced by the hormones administered. The activities of NADP+-linked malate dehydrogenase, citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase, although lower in tumors than in normal glands, were not different in HD as compared to HI tumors. Whereas the predominating lipids synthesized in normal glands were triglycerides, phospholipids comprised about half of the lipid synthesized in the tumors, with no difference between HD and HI tumors. The conversion of D-[U-14C]glucose to 14CO2 was higher in HD tumors than in HI tumors but increased in HI tumors treated with hormones in vivo. By a comparison of the 14CO2 produced from D-[1-14C]glucose and from D-[6-14C]glucose in the presence and absence of an electron acceptor (methylene blue), it was demonstrated that regeneration of NADP+ from NADPH was a rate-limiting step for the pentose phosphate pathway in the tumors. Hence, while differences in the lipid metabolism can be demonstrated between HD and HI GR mouse mammary tumors, some of the changes are due to the hormone treatment rather than to a specific alteration in the tumor itself.

Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP + cycling at high efficiency

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP + was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD + oxidoreductase, EC 1.1.1.176), and the regeneration of NADP + was obtained through the glutamate dehydrogenase ( l-glutamate:NADP + oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained ∼70% of the initial activity after 2 months of continuous use.

Enzymatic preparation of 12‐ketochenodeoxycholic acid with NADP regeneration

Biotechnology and BioengineeringVolume 26, Issue 5 p. 560-563 Communications to The EditorFree Access Enzymatic preparation of 12-ketochenodeoxycholic acid with NADP regeneration G. Carrea, G. Carrea Istituto di Chimica degli Ormoni, C.N.R., Via Mario Bianco 9, 20131 Milano, ItalySearch for more papers by this authorR. Bovara, R. Bovara Istituto di Chimica degli Ormoni, C.N.R., Via Mario Bianco 9, 20131 Milano, ItalySearch for more papers by this authorP. Cremonesi, P. Cremonesi Italfarmaco, Viale F. Testi 330, Milano, ItalySearch for more papers by this authorR. Lodi, R. Lodi Centro per lo Studio Tecnologico, Bromatologico e Microbiologico del Latte, C.N.R., Via Celoria 2, 20131 Milano, ItalySearch for more papers by this author G. Carrea, G. Carrea Istituto di Chimica degli Ormoni, C.N.R., Via Mario Bianco 9, 20131 Milano, ItalySearch for more papers by this authorR. Bovara, R. Bovara Istituto di Chimica degli Ormoni, C.N.R., Via Mario Bianco 9, 20131 Milano, ItalySearch for more papers by this authorP. Cremonesi, P. Cremonesi Italfarmaco, Viale F. Testi 330, Milano, ItalySearch for more papers by this authorR. Lodi, R. Lodi Centro per lo Studio Tecnologico, Bromatologico e Microbiologico del Latte, C.N.R., Via Celoria 2, 20131 Milano, ItalySearch for more papers by this author First published: May 1984 https://doi.org/10.1002/bit.260260526Citations: 33AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume26, Issue5May 1984Pages 560-563 ReferencesRelatedInformation

Crystallization and characterization of NADP-dependent D-glucose dehydrogenase from Gluconobacter suboxydans.

NADP-Dependent D-glucose dehydrogenase (EC 1.1.1.47) was crystallized for the first time from cytosol fraction of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A-50 and affinity chromatography by blue-dextran Sepharose 4B. The enzyme was purified about 1, 800-fold with an overall yield of 30%. Crystalline enzyme preparation was homogeneous in disc gel electrophoresis and analytical ultracentrifugation. The enzyme was highly specific for NADP and completely inactive with NAD. NADPH yielded in D-glucose oxidation to D-glucono-δ-lactone was reoxidized to NADP by the old yellow enzyme which existed in the same cytosol fraction of the organism. Cyclic regeneration of NADP occurred smoothly in the presence of D-glucose dehydrogenase, old yellow enzyme and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.