Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.     

A novel aspect of NADPH production in ageing Penicillium chrysogenum

NADPH is involved in many basically important anabolic processes. For a long time, pentose phosphate pathway (PPS) was regarded as the most important source of NADPH in fungi. Here we present evidence of a metabolic switch to an alternative NADPH-producing pathway in ageing Penicillium chrysogenum cultures, which involves NADP+ -specific isocitrate dehydrogenase (NADP+ -ID) rather than PPS enzymes. Considering the main biochemical functions of NADPH, we propose that NADP+ -ID could have deep impact on many physiological processes switched on glucose deprivation including proteinase production or penicillin biosynthesis. We also demonstrate that although the alternative pathway was inferior to PPS when the fungus was grown on well-utilisable carbon sources yet it could have an important role in fatty acid biosynthesis as well as in the maintenance of high intracellular NADPH/NADP+ ratios.     

The role of malic enzyme in the malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

Mitochondria isolated from human term placenta were able to form citrate from malate as the only added substrate. While mitochondria were incubated in the presence of Mn2+ the citrate formation was stimulated significantly both by NAD+ and NADP+ and was inhibited by hydroxymalonate, arsenite, butylmalonate and rotenone. It is concluded that NAD(P)-linked malic enzyme is involved in the conversion of malate to citrate in these mitochondria. It has also been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate was stimulated by NAD+ and NADP+ and inhibited by arsenite and fluorocitrate. This suggests that the stimulation by malate of progesterone biosynthesis depends not only on the generation of NADPH by NAD(P)-linked malic enzyme, but also on NADPH formed during further metabolism of pyruvate to isocitrate which is in turn efficiently oxidized by NADP+-linked isocitrate dehydrogenase.     

Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens

The fate of all aerobic organisms is dependent on the varying intracellular concentrations of NADH and NADPH. The former is the primary ingredient that fuels ATP production via oxidative phosphorylation, while the latter helps maintain the reductive environment necessary for this process and other cellular activities. In this study we demonstrate a metabolic network promoting NADPH production and limiting NADH synthesis as a consequence of an oxidative insult. The activity and expression of glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-isocitrate dehydrogenase, the main generators of NADPH, were markedly increased during oxidative challenge. On the other hand, numerous tricarboxylic acid cycle enzymes that supply the bulk of intracellular NADH were significantly downregulated. These metabolic pathways were further modulated by NAD(+) kinase (NADK) and NADP(+) phosphatase (NADPase), enzymes known to regulate the levels of NAD(+) and NADP(+). While in menadione-challenged cells, the former enzyme was upregulated, the phosphatase activity was markedly increased in control cells. Thus, NADK and NADPase play a pivotal role in controlling the cross talk between metabolic networks that produce NADH and NADPH and are integral components of the mechanism involved in fending off oxidative stress.     

Regulation of C4 photosynthesis: regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.     

Physiological role of yeasts NAD(P)+ and NADP+-linked aldehyde dehydrogenases.

The activity of NAD+ and NADP+-linked aldehyde dehydrogenases has been investigated in yeast cells grown under different conditions. As occurs in other dehydrogenase reactions the NAD(P)+-linked enzyme was strongly repressed in all hypoxic conditions; nervetheless, the NADP+-linked enzyme was active. The results suggest that the NAD(P)+ aldehyde dehydrogenase is involved in the oxidation of ethanol to acetyl-CoA, and that when the pyruvate dehydrogenase complex is repressed the NADP+-linked aldehyde dehydrogenase is operative as an alternative pathway from pyruvate to acetyl-CoA: pyruvate leads to acetaldehyde leads to acetate leads to acetyl-Coa. In these conditions the supply of NADPH is advantageous to the cellular economy for biosynthetic purposes. Short term adaptation experiments suggest that the regulation of the levels of the aldehyde dehydrogenase-NAD(P)+ takes place by the de novo synthesis of the enzyme.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Efficient regeneration of NADPH using an engineered phosphite dehydrogenase

The in situ regeneration of reduced nicotinamide cofactors (NAD(P)H) is necessary for practical synthesis of many important chemicals. Here, we report the engineering of a highly stable and active mutant phosphite dehydrogenase (12x-A176R PTDH) from Pseudomonas stutzeri and evaluation of its potential as an effective NADPH regeneration system in an enzyme membrane reactor. Two practically important enzymatic reactions including xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis were used as model systems, and the mutant PTDH was directly compared to the commercially available NADP(+)-specific Pseudomonas sp. 101 formate dehydrogenase (mut Pse-FDH) that is widely used for NADPH regeneration. In both model reactions, the two regeneration enzymes showed similar rates of enzyme activity loss; however, the mutant PTDH showed higher substrate conversion and higher total turnover numbers for NADP(+) than mut Pse-FDH. The space-time yields of the product with the mutant PTDH were also up to fourfold higher than those with mut Pse-FDH. In particular, a space-time yield of 230 g L(-1) d(-1) xylitol was obtained with the mutant PTDH using a charged nanofiltration membrane, representing the highest productivity compared to other existing biological processes for xylitol synthesis based on yeast D-xylose converting strains or similar in vitro enzyme membrane reactor systems.     

Formate as an auxiliary substrate for glucose-limited cultivation of Penicillium chrysogenum: impact on penicillin G production and biomass yield

Production of beta-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol.mol(-1), an increasing rate of formate oxidation via a cytosolic NAD(+)-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol.mol(-1), the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as beta-lactams.     

Control of hepatic nuclear superoxide production by glucose 6-phosphate dehydrogenase and NADPH oxidase-4

Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O(2)(·-)); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22(phox), and p47(phox)) contribute to nuclear O(2)(·-) production in isolated hepatic nuclei. Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O(2)(·-) production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O(2)(·-) predominantly localized to the perinuclear space. Interestingly, NADP(+) and G6P also induced nuclear O(2)(·-) production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O(2)(·-) in response to NADP(+) and G6P. NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O(2)(·-) production by NOX4.     

Growth characteristics and metabolic flux analysis of Candida milleri

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.     

Yeast-like cell formation and glutathione metabolism in autolysing cultures of Penicillium chrysogenum

The bulk formation of yeast-like (arthrospore-like) cells were typical in carbon-depleted submerged cultures of the high beta-lactam producer Penicillium chrysogenum NCAIM 00237 strain independently of the nitrogen-content of the culture medium. This morphogenetic switch was still quite common in carbon-starving cultures of the low-penicillin-producer strain P. chrysogenum ATCC 28089 (Wis 54-1255) when the nitrogen-content of the medium was low but was a very rare event in wild-type P. chrysogenum cultures. The mycelium-->yeast-like cell transition correlated well with a relatively high glutathione concentration and a reductive glutathione/glutathione disulfite (GSH/GSSG) redox balance in autolysing cultures, which was a consequence of industrial strain development. Paradoxically, the development of high beta-lactam productivity resulted in a high intracellular GSH level and, concomitantly, in an increased y-glutamyltranspeptidase (i.e. GSH-decomposing) activity in the autolytic phase of growth of P. chrysogenum NCAIM 00237. The hypothesized causal connection between GSH metabolism and cell morphology, if verified, may help us in future metabolic engineering of industrially important filamentous fungi.     

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.     

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.     

The B' helix determines cytochrome P450nor specificity for the electron donors NADH and NADPH

Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here, we report that oxidized pyridine nucleotides (NAD(+) and NADP(+)) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor, a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD(+) induced a type I spectral change in Fnor, whereas NADP(+) induced a reverse type I spectral change, although the K(d) values for both were comparable. In contrast, NADP(+) as well as NAD(+) caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B' helix region of Tnor ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). A single mutation (S75G) significantly improved the NADPH- dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B' helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.     

Purification and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe.

6-Phosphogluconate dehydrogenase is the pivotal enzyme that links the gluconate route and the oxidative phase of the pentose phosphate pathway in Schizosaccharomyces pombe. The enzyme differs from the known 6-phosphogluconate dehydrogenases of other sources in that the Schizosaccharomyces enzyme is tetrameric having a subunit mass of 38 kDa, that it requires NADP+ obligatorily for activity, and that it can be activated by divalent metal ions such as Co2+ and Mn2+. Steady-state kinetic studies were undertaken. Initial rate and product inhibition results suggest that 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe catalyzes NADP(+)-linked oxidative decarboxylation of 6-phosphogluconate by an equilibrium random mechanism with two independent binding sites, namely one site for the nicotinamide coenzyme, NADP+/NADPH, and another site for 6-phosphogluconate-D-ribulose-5-phosphate and for CO2. Studies of pH dependence implicated a basic residue with a pK value of 7.4 in the binding of 6-phosphogluconate and an acidic residue with a pK value of 6.7 in the cation-mediated interaction of NADP+ with the enzyme.