Inhibition of growth by erythritol catabolism in Brucella abortus.

The growth of Brucella abortus (US-19) in a complex tryptose-yeast extract medium containing D-glucose is inhibited by 10 mM erythritol. The enzymes of the erythritol pathway, except for D-erythrulose 1-phosphate dehydrogenase (D-glycero-2-tetrulose 1-phosphate:nicotinamide adenine dinucleotide (NAD+) 4-oxidoreductase) were detected in the soluble and membrane fractions of cell extracts. Glucose catabolism by cell extracts was inhibited by erythritol, whereas, phosphorylated intermediates of the hexose monophosphate pathway were converted to pyruvic acid with oxygen consumption. Erythritol kinase (EC 2.7.1.27; adenosine 5'-triphosphate (ATP): erythritol 1-phosphotransferase) was found to be eightfold higher in activity than the hexokinase in cell extracts. In vivo, ATP is apparently consumed with the accumulation of D-erythrulose 1-phosphate (D-glycero-2-tetrulose 1-phosphate) and no substrate level phosphorylation. ATP levels dropped 10-fold in 30 min after addition of erythritol to log phase cells in tryptose-yeast extract medium with D-glucose as the carbon source. These data suggest bacteriostasis in the presence of erythritol results from the ATP drain caused by erythritol kinase.     

Control of erythritol dehydrogenase in Schizophyllum commune

Summary An NAD-dependent erythritol dehydrogenase was detected in cell-extracts of basidiospore germinants of Schizophyllum commune following culture on either meso-erythritol or glycerol as sole carbon sources. Induction of erythritol dehydrogenase was also observed in purely vegetative mycelium (str. 845 or str. 699). Erythritol dehydrogenase was not observed in ungerminated basidiospores or germinants which arose on d-glucose, d-mannitol, sorbitol, ribitol, xylitol, d-arabitol or l-arabitol. NAD-coupled polyol dehydrogenases for all the latter sugar alcohols were observed in ungerminated basidiospores, germinants, and vegetative mycelium of S. commune cultured on d-glucose. Basidiospore germination on d-glucose plus meso-erythritol led to a 90% decrease in erythritol dehydrogenase and the specific activity of ribitol dehydrogenase was directly comparable to that seen in d-glucose germinants. Storage experiments of crude extracts of meso-erythritol germinants indicated differential enzyme decay of dehydrogenases for d-mannitol, sorbitol and erythritol while the respective enzymes could be further distinguished by heat-stability as well as preferential utilization of analogues of NAD. DEAE-cellulose column chromatography led to separation of sorbitol dehydrogenase which was also active with xylitol, erythritol dehydrogenase, and mannitol dehydrogenase which was also active with d-arabitol.     

Enzymes of Carbohydrate Metabolism in Thiobacillus species

A study was made of enzymes of carbohydrate metabolism in representative thiobacilli grown with and without glucose. The data show that Thiobacillus perometabolis possesses an inducible Entner-Doudoroff pathway and is thus similar to T. intermedius and T. ferrooxidans. T. novellus lacks this pathway. Instead, a non-cyclic pentose phosphate pathway along with the Krebs cycle is apparently the major route of glucose dissimilation in this organism. Glucose does not support or stimulate the growth of strains of T. neapolitanus, T. thioparus, and T. thiooxidans examined, nor does its presence in the growth medium greatly influence their enzymatic constitution. These obligately chemolithotrophic thiobacilli do not possess the Entner-Doudoroff pathway. Their nicotinamide adenine dinucleotide (NAD)-linked isocitrate dehydrogenase activity predominates over their nicotinamide adenine dinucleotide phosphate (NADP)-linked activity; the converse is true for the other thiobacilli. The data suggest that NAD-linked isocitrate dehydrogenase activity in thiobacilli is involved in biosynthetic reactions.     

Regulation of glutamate dehydrogenases during morphogenesis of Schizophyllum commune

The specific activities of two glutamate dehydrogenases (GDH), one requiring nicotinamide adenine dinucleotide (NAD) and the other specific for nicotinamide adenine dinucleotide phosphate (NADP), varied during growth of Schizophyllum commune as a function of the stage of the life cycle and the exogenous nitrogen source. During basidiospore germination on either glucose-NH(3) or glucose-glutamate medium, NADP-GDH increased six- to eightfold in specific activity, whereas NAD-GDH was depressed. During dikaryotic mycelial growth on either nitrogen source, the two GDH increased in a 1:1 ratio, whereas, during homokaryotic mycelial growth on glucose-NH(3), NADP-GDH activity was depressed and NAD-GDH increased six- to eightfold. Homokaryotic mycelium cultured on glucose-glutamate medium yielded high NADP-GDH activities and normal NAD-GDH activities. Intracellular NH(3) concentration and NADP-GDH activities were inversely related during spore germination and homokaryotic mycelium growth, whereas guanosine-5'-triphosphate (GTP) and l-glutamine specifically inhibited NAD- and NADP-GDH respectively in vitro. GTP inhibition was shown in extracts from cells at all stages of the life cycle. Basidiospore germling extracts contained an NADP-GDH essentially resistant to l-glutamine inhibition.     

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.     

Formation and dissimilation of oxalacetate and pyruvate Pseudomonas citronellolis grown on noncarbohydrate substrates.

Metabolism of lactate as a carbon source by Pseudomonas citronellolis occurred via a nicotinamide adenine dinucleotide (NAD)-independent L-lactate dehydrogenase, which was present in cells grown on DL-lactate but was not present in cells grown on acetate, aspartate, citrate, glucose, glutamate, or malate. The cells also possessed a constitutive, NAD-independent malate dehydrogenase instead of the conventional NAD-dependent malate dehydrogenase instead of the conventional NAD-dependent enzyme in the tricarboxylic acid cycle. Both enzymes were particulate and used dichlorophenolindo-phenol or oxygen as an electron acceptor. In acetate-grown cells, the activity of pyruvate dehydrogenase and NAD phosphate-linked malate enzyme decreased, cells grown on glucose or lactate. This was consistent with the need to maintain a supply of oxalacetate for metabolism of acetate via the tricarboxylic acid cycle. Changes in enzyme activities suggest that gluconeogenesis from noncarbohydrate carbon sources occurs via the malate enzyme (when oxalacetate decarboxylase is inhibited) or a combination of the NAD-independent malate dehydrogenase and oxalacetate decarboxylase.     

In Yarrowia lipolytica erythritol catabolism ends with erythrose phosphate

In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four‐carbon sugar alcohol, from erythrose‐P, an intermediate of the pentose phosphate pathway. Under non‐stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose‐P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose‐1P isomerase that converts erythrulose‐1P into erythrulose‐4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.     

Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which is derived from NAD, have important roles as a redox carriers in metabolism. A combination of de novo and salvage pathways contribute to the biosynthesis of NAD in all organisms. The pathways and enzymes of the NAD salvage pathway in yeast and animals, which diverge at nicotinamide, have been extensively studied. Yeast cells convert nicotinamide to nicotinic acid, while mammals lack the enzyme nicotinamidase and instead convert nicotinamide to nicotinamide mononucleotide. Here we show that Arabidopsis thaliana gene At2g22570 encodes a nicotinamidase, which is expressed in all tissues, with the highest levels observed in roots and stems. The 244-residue protein, designated AtNIC1, converts nicotinamide to nicotinic acid and has a Km value of 118 +/- 17 microM and a Kcat value of 0.93 +/- 0.13 sec(-1). Plants homozygous for a null AtNIC1 allele, nic1-1, have lower levels of NAD and NADP under normal growth conditions, indicating that AtNIC1 participates in a yeast-type NAD salvage pathway. Mutant plants also exhibit hypersensitivity to treatments of abscisic acid and NaCl, which is correlated with their inability to increase the cellular levels of NAD(H) under these growth conditions, as occurs in wild-type plants. We also show that the growth of the roots of wild-type but not nic1-1 mutant plants is inhibited and distorted by nicotinamide.     

Identification and characterization of EYK1 , a key gene for erythritol catabolism in Yarrowia lipolytica

Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest.     

Regulation of alanine dehydrogenase in Bacillus (licheniformis)

Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent l-alanine dehydrogenase (ADH) (l-alanine: NAD oxidoreductase, EC 1.4.1.1). High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when l-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logarithmic-phase growth on l-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM l-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, l-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. licheniformis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of l-alanine to pyruvate during vegetative growth on l-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results.     

Evidence for two species of glutamate dehydrogenases in Thiobacillus novellus

When grown autotrophically in a thiosulfate-mineral salts medium, cells of the facultative chemoautotrophic bacterium, Thiobacillus novellus, produced two distinct glutamate dehydrogenases, one specific for nicotinamide adenine dinucleotide phosphate (NADP) and the other specific for nicotinamide adenine dinucleotide (NAD). When glutamate was supplied exogenously as the sole carbon source, the NAD-specific glutamate dehydrogenase was fully induced. Lower levels of the enzyme were found in bacteria grown in l-arginine, l-alanine, glucose, glycerol, lactate, citrate, or succinate. Arginine, histidine, and aspartate, on the other hand, caused a marked repression of the NADP-specific glutamate dehydrogenase activity. The NAD-dependent glutamate dehydrogenase was allosteric. Adenosine-5'-monophosphate and adenosine-5'-diphosphate acted as positive effectors. Both glutamate dehydrogenases were purified about 250-fold and were shown to be distinct protein with different physical properties.     

Pyridine Nucleotide-Dependent Glucose Dehydrogenase Activity in Blue-Green Algae

Pyridine nucleotide-dependent glucose dehydrogenase activity (GPND) is described for the first time in cell-free extracts of certain blue-green algae. When glucose is added to these crude cell extracts, nicotinamide adenine dinucleotide phosphate is reduced at twice the rate as nicotinamide adenine dinucleotide; but evidence suggests that this activity is due to a single enzyme. The distribution and level of GPND in selected blue-green algae correlates with the heterotrophic potential of each species. In all blue-green algae where GPND was detected, O(2) uptake coupled to the GPND reaction was also observed. Both GPND and O(2) uptake apparently occur in the soluble fraction of the cell. An essential role for GPND in the heterotrophic metabolism of blue-green algae is postulated.     

Heterotrophic Metabolism of the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase and the enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase (assayed together), are induced during heterotrophic growth of Thiobacillus ferrooxidans on an iron-glucose-supplemented medium or on glucose alone. By contrast, autotrophic cells (iron-grown) contain low levels of these enzymes. Fructose 1, 6-diphosphate aldolase, an enzyme of the Embden-Meyerhof pathway, is present at low levels irrespective of the growth medium, suggesting that this enzyme is not involved in energy-yielding reactions but merely provides intermediates for biosynthesis. The Entner-Doudoroff and pentose-phosphate pathways are the principle means through which glucose is dissimilated and is presumed to be concerned with energy production. Isotopic studies showed that a high rate of CO(2) formation from specifically labeled glucose came from carbon atoms 1 and 4. An unexpectedly high rate of evolution of CO(2) also came from carbon 6, suggesting that the triose phosphate formed during glucose breakdown and specifically as a result of 2-keto-3-deoxy-6-phosphogluconate aldolase activity, was metabolized via some unorthodox metabolic route. Cells grown in the iron-supplemented and glucose-salts media have a complete tricarboxylic acid cycle, whereas autotrophically grown T. ferrooxidans lacked both alpha-ketoglutarate dehydrogenase and reduced nicotinamide adenine dinucleotide oxidase. Two isocitrate dehydrogenases [nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) specific] were present. NAD-linked enzyme was constitutive, whereas the NADP-linked enzyme was induced upon adaptation of autotrophic cells to heterotrophic growth.     

Enzymatic Synthesis of l-Carnitine by Reduction of an Achiral Precursor: the Problem of Reduced Nicotinamide Adenine Dinucleotide Recycling

Synthesis of l-carnitine has been carried out by the enzymatic reduction of the carbonyl group of the achiral precursor 3-dehydrocarnitine with the oxidized nicotinamide adenine dinucleotide-linked carnitine dehydrogenase. Various enzymatic or chemical systems have been tested to regenerate the reduced nicotinamide adenine dinucleotide oxidized in the reduction of 3-dehydrocarnitine. Because of the instability of this compound in aqueous solutions, it was added by continuous feeding as a rate-limiting constituent in the reaction mixture. Under these conditions, conversion yields of 95% were achieved with the glucose plus glucose dehydrogenase system. A total number of 530 reduced nicotinamide adenine dinucleotide recyclings was obtained with this system for a production of 45 g of l-carnitine per liter. The stabilities of the oxidized nicotinamide adenine dinucleotide and the reduced nicotinamide adenine dinucleotide have been determined at various pH values. In view of these results, several possible strategies for enzymatic syntheses with the reduced nicotinamide adenine dinucleotide as a regenerable coenzyme are discussed.     

Did primitive microorganisms use nonhem iron proteins in place of NAD/P?

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are of universal occurrence in living organisms and play a central role in coupling oxidative with reductive reactions. However, the evidence that the origin and early evolution of life occurred at high temperatures (>95°C) is now strong, and at these temperatures some modern metabolites, including both the reduced and oxidized forms of these coenzymes, are unstable. We believe there is good evidence that indicates that in the most primitive organisms nonhem iron proteins carried out many or all of the functions of NAD/P(H). This has important implications for the way in which investigations of archaebacterial metabolism are conducted.Abbreviations NAD/P(H)a Oxidised and reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate     

Observations on the Regulation of Glutamate Dehydrogenase Activity in Coprinus lagopus

Extracts of the mycelium of Coprinus lagopus (sensu Buller)contain two glutamate dehydro-genases with different optimumpH values. One is assayed with nicotinamide adenine dinucleotide(NAD-GDH) and the other with nicotinamide adenine dinucleotidephosphate (NADP-GDH). Changes in specific activity of the enzymeswere investigated during the growth of both a monokaryon (H9)and a dikaryon (H9 x TC) in different media and after the transferof mycelium from one growth medium into another. In the lattercase the magnitude of the changes in enzyme activity could bealtered by modification of either the carbon or the nitrogensource in the transfer medium. It is concluded from the resultsobtained that neither glutamate nor the ammonium ion seems toregulate directly the synthesis of either enzyme. However, someof the results are in accordance with the view that a productof glucose metabolism represses the synthesis of the NAD-GDHand derepresses or induces that of the NADP-CDH and evidencethat this regulator is 2-oxoglutarate was obtained. It is alsoconcluded that the complete system of regulation must involvemore than one molecule.     

Use of Nicotinamide Adenine Dinucleotide (NAD)-Dependent Glucose-6-Phosphate Dehydrogenase in Enzyme Staining Procedures

Substitution of nicotinamide adenine dinucleotide dependent glucose-6-phosphate dehydrogenase for the nicotinamide adenine dinucleotide phosphate dependent enzyme has produced identical results in a number of enzyme-linked electrophoretic staining procedures. This substitution significantly reduces the cost of staining for adenylate kinase, creatine kinase, glucosephosphate isomerase, mannosephosphate isomerase, phosphoglucomutase, and pyruvate kinase activity by utilizing NAD rather than the more expensive NADP.     

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.     

Regulation of Staphylococcus aureus Lactate Dehydrogenase

The effect of growth conditions on the specific activity of nicotinamide adenine dinucleotide (NAD)-linked lactate dehydrogenase (LDH) in extracts of Staphylococcus aureus strain SG 511A was examined. Kinetic and electrophoretic experiments, with extracts prepared from aerobically and anaerobically grown cells, provided evidence for only one physiologically significant enzyme. The aerobic level of NAD-linked LDH of S. aureus remained constant and was independent of the carbon source. In contrast, the level of LDH produced in an anaerobic environment was variable and was dependent on the carbon source. Growth anaerobically on pyruvate, as the sole fermentable carbon source, resulted in a maximal level of LDH activity, a value about eightfold greater than the aerobic level. Anaerobic growth either on pyruvate plus glucose or on glucose alone, however, resulted in approximately a threefold decrease in this maximum. Experiments with a heme-requiring auxotroph derived from S. aureus demonstrated that the aerobic level of LDH activity was dependent on a functional respiratory chain.     

Carbohydrate Metabolism During Morphogenesis of Coprinus lagopus (sensu Buller)

The occurrence and properties of enzymes of carbohydrate metabolism were studied during dikaryotic fruiting of the mushroom Coprinus lagopus. Enzymes of hexose monophosphate catabolism, sugar alcohol (polyol) dehydrogenases (DH), and trehalase occurred throughout development. The ratio of xylitol DH to sorbitol DH was greater than unity in both monokaryotic mycelium and dikaryotic fruit body caps, whereas this ratio decreased in the stipe (stalk) tissue. Xylitol DH and sorbitol DH were both dependent upon nicotinamide adenine dinucleotide (NAD) and showed maximal activity at pH 9. Two separate enzymes were suspected on the basis of preferential utilization of the NAD analogue, thionicotinamide-NAD, by xylitol DH, and this feature was consistent throughout development. An appraisal of the carbohydrate pool revealed trehalose and glucose, with the former predominant in the stipe and the latter in excess in the cap of dikaryotic fruit bodies. Trehalase activity in dialyzed enzyme extracts showed pH optima at acid and alkaline pH levels in monokaryotic mycelium, dikaryotic stipes, and cap tissues.