Thiosulfate- and Sulfide-Dependent Pyridine Nucleotide Reduction and Gluconeogenesis in Intact Thiobacillus neapolitanus

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.     

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.     

Catabolism of L-tyrosine in Trichosporon cutaneum.

Protocatechuic acid was a catabolite in the degradation of L-tyrosine by Trichosporon cutaneum. Intact cells oxidized to completion various compounds proposed as intermediates in this conversion, but they did not readily oxidize catabolites of the homogentisate and homoprotocatechuate metabolic pathways, which are known to function in other organisms. Cell extracts converted tyrosine first to 4-hydroxycinnamic acid and then to 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid. The proposed hydration product of 4-hydroxycinnamic acid, namely, beta-(4-hydroxyphenyl)-hydracrylic acid, was synthesized chemically, and its enzymatic degradation to 4-hydroxybenzaldehyde was shown to be dependent upon additions of adenosine triphosphate and coenzyme A. The hydroxylase that attacked 4-hydroxybenzoate showed a specific requirement for reduced nicotinamide adenine dinucleotide phosphate. Protocatechuate, the product of this reaction, was oxidized by cell extracts supplemented with reduced nicotinamide adenine dinucleotide or, less effectively, with reduced nicotinamide adenine dinucleotide phosphate, but these extracts contained no ring fission dioxygenase for protocatechuate. Evidence is presented that the principal hydroxylation product of protocatechuate was hydroxyquinol, the benzene nucleus of which was cleaved oxidatively to give maleylacetic acid.     

A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD)

Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.     

Aldohexuronic acid catabolism by a soil Aeromonas

Bacteria which utilize mannuronic acid as an energy source were isolated from nature. One of the organisms, identified as a member of the genus Aeromonas, used glucuronate, galacturonate, and mannuronate as the sole source of carbon and energy. Glucuronate- and galacturonate-grown resting cells oxidized both glucuronate and galacturonate rapidly, but mannuronate slowly. Mannuronate-grown cells oxidized all three rapidly, with the rate of mannuronate utilization somewhat lower. Cell-free extracts from glucuronate-, galacturonate-, and mannuronate-grown Aeromonas C11-2B contained glucuronate and galacturonate isomerases, fructuronate, tagaturonate, and mannuronate reductases, and mannonate and altronate dehydratases, with the exception of glucuronate-grown cells which lacked altronate dehydratase. Thus, the pathway for glucuronate and galacturonate catabolism for Aeromonas was identical to Escherichia coli. Glucuronate and galacturonate were isomerized to d-fructuronate and d-tagaturonate which were then reduced by reduced nicotinamide adenine dinucleotide to d-mannonate and d-altronate, respectively. The hexonic acids were dehydrated to 2-keto-3-deoxy gluconate which was phosphorylated by adenosine triphosphate to 2-keto-3-deoxy-6-phospho gluconate. The latter was then cleaved to pyruvate and glyceraldehyde-3-phosphate. Mannuronate was reduced directly to d-mannonate by a reduced nicotinamide adenine dinucleotide phosphate-linked oxidoreductase. d-Mannonate was then further broken down as in the glucuronate pathway. The mannuronate reducing enzyme, for which the name d-mannonate:nicotinamide adenine dinucleotide (phosphate) oxidoreductase (d-mannuronate-forming) was proposed, was shown to be distinct from altronate and mannoate oxidoreductases. This is the first report of a bacterial oxidoreductase which reduces an aldohexuronic acid to a hexonic acid. The enzyme should prove to be a useful analytical tool for determining mannuronate in the presence of other uronic acids.     

Plasmid specifying total degradation of 3-chlorobenzoate by a modified ortho pathway.

We purified lipoamide dehydrogenase from cells of Pseudomonas putida PpG2 grown on glucose (LPD-glu) and lipoamide dehydrogenase from cells grown on valine (LPD-val), which contained branched-chain keto acid dehydrogenase. LPD-glu had a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and LPD-val had a molecular weight of 49,000. The pH optimum for LPD-glu for reduced nicotinamide adenine dinucleotide oxidation was 7.4, compared with pH 6.5 for LPD-val. When oxidized nicotinamide adenine dinucleotide was included in the assay mixture, the pH optima were 7.1 and 5.7, respectively. There was also a difference in pH optima between the two enzymes for oxidized nicotinamide adenine dinucleotide reduction, but the Michaelis constants and maximum velocities were similar. A purified preparation of branched-chain keto acid dehydrogenase, which was deficient in lipoamide dehydrogenase, was stimulated 10-fold by LPD-val but not by LPD-glu, which suggested that the branched-chain keto acid dehydrogenase of P. putida has a specific requirement for LPD-val. In contrast, a partially purified preparation of 2-ketoglutarate dehydrogenase that was deficient in lipoamide dehydrogenase was stimulated by LPD-glu but not by LPD-val, indicating that this complex has a specific requirement of LPD-glu.     

Single-sample preparation for simultaneous cellular redox and energy state determination

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.     

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.     

High-performance ion-exchange separation of oxidized and reduced nicotinamide adenine dinucleotides

High-performance anion-exchange chromatography of oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) on a Pharmacia Mono Q anion-exchange column is reported. Microgram quantities of all four nucleotides can be separated at pH 7.7 in approximately 20 min. For preparative purposes, greater than 7 mg of NADH can be purified in a single injection, and the peak fractions have an A260 of greater than 80 OD units with an A260/A340 ratio of 2.25.     

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     

Metabolism of l-Malate and d-Malate by a Species of Pseudomonas

Extracts of a fluorescent species of Pseudomonas grown with m-cresol, degrade gentisic acid without isomerization of the ring-fission compound, maleylpyruvate, to give eventually d-malate and pyruvate. d-Malate is also a growth substrate. l-Malate but not d-malate is oxidized by a particulate enzyme not requiring nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). NAD- or NADP-linked malate dehydrogenases are absent but cells contain an NADP-dependent l-malic enzyme. Exposure of cells to exogenous d-malate induces an NAD-dependent d-malic enzyme, not present when d-malate is formed endogenously. Succinate- or m-cresol-grown cells, containing no d-malic enzyme, rapidly oxidize d-malate in the presence of chloramphenicol at a concentration suffient to inhibit protein synthesis. An NADP-dependent cell-free system, prepared from succinate-grown cells which oxidized d-malate, is described.     

The Regulatory Properties of Purified Phaseolus aureus Sucrose Synthetase

Phaseolus aureus sucrose synthetase, purified to homogeneity, was assayed in the presence of a variety of biological compounds to test for possible regulatory effectors. The oxidized form of nicotinamide adenine dinucleotide phosphate, as well as indoleacetic acid, gibberellic acid, and pyrophosphate were found to activate the forward reaction (sucrose degradation) and inhibit the reverse reaction (sucrose synthesis). The reduced form of nicotinamide adenine dinucleotide phosphate antagonizes the effect of the oxidized form. Fructose 1-phosphate and divalent cations inhibit the forward and activate the reverse reaction. Pyrophosphate and fructose 1-phosphate are effective only in the presence of magnesium chloride. Uridine triphosphate inhibits both the forward and reverse reactions. All effectors except gibberellic acid are active only in the millimolar range of concentrations; maximal stimulation for any effector is approximately 2-fold. The effects of combinations of effectors are roughly additive. Using pyrophosphate in the presence of magnesium chloride as an effector, results of kinetic studies offer a model by which an effector can activate an enzymatic reaction in one direction and inhibit in the reverse direction.     

Pathway of n-Alkane Oxidation in Cladosporium resinae

Pathways of initial oxidation of n-alkanes were examined in two strains of Cladosporium resinae. Cells grow on dodecane and hexadecane and their primary alcohol and monoic acid derivatives. The homologous aldehydes do not support growth but are oxidized by intact cells and by cell-free preparations. Hexane and its derivatives support little or no growth, but cell extracts oxidize hexane, hexanol, and hexanal. Alkane oxidation by extracts is stimulated by reduced nicotinamide adenine dinucleotide (phosphate). Alcohol and aldehyde oxidation are stimulated by nicotinamide adenine dinucleotide (phosphate), and reduced coenzymes accumulate in the presence of cyanide or azide. Extracts supplied with (14)C-hexadecane convert it to the alcohol, aldehyde, and acid. Therefore, the major pathway for initial oxidation of n-alkanes is via the primary alcohol, aldehyde, and monoic acid, and the system can act on short-, intermediate-, and long-chain substrates. Thus, filamentous fungi appear to oxidize n-alkanes by pathways similar to those used by bacteria and yeasts.     

A nuclear magnetic resonance-based functional assay for nicotinamide adenine dinucleotide synthetase

Nicotinamide adenine dinucleotide synthetase (NadE) is an essential enzyme for bacterial pathogens and is thus a promising antibacterial target. It catalyzes the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. Changes in chemical shifts that occur in the nicotinic acid ring as it is converted to nicotinamide can be used for monitoring the reaction. A robust nuclear magnetic resonance-based activity assay was developed using robotically controlled reaction initiation and quenching. The single-enzyme assay has less potential for false positives compared to a coupled activity assay and is especially well suited to the high concentration of compounds in fragment screens. The assay has been used to screen fragment libraries for NadE inhibitors.     

Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.

We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

An enzyme-coupled assay for glyoxylic acid

Herein, we present a new enzyme-linked spectrophotometric assay for glyoxylate that detects glyoxylate via the formation of an intensely colored formazan. This glyoxylate-specific assay is reliant upon the enzymatic conversion of glyoxylate to oxaloacetate coupled to the reduction of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide (NADH). The NADH-dependent reduction of a tetrazolium to the formazan enables the measurement of nanomole quantities of glyoxylate in an assay that is amenable to high-throughput screening methods. Assay validation was accomplished using two methods for glyoxylate generation, the base-catalyzed N-dealkylation of alpha-hydroxyhippurate to benzamide and glyoxylate and the oxidative cleavage of the glycyl Calpha-N bond in N-benzoylglycine (hippurate) by peptidylglycine alpha-amidating monooxygenase to again yield benzamide and glyoxylate. For both reactions, analysis of benzamide produced by reverse-phase high-performance liquid chromatography compared with glyoxylate measured using our glyoxylate assay showed a 1:1 molar ratio of benzamide to glyoxylate. These results indicate that the enzyme-linked spectrophotometric assay can quantitatively measure submicromole quantities of glyoxylate.     

Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.     

Two succinic semialdehyde dehydrogenases are induced when Escherichia coli K-12 Is grown on gamma-aminobutyrate.

When Escherichia coli K-12 was grown on gamma-aminobutyrate, a second succinic semialdehyde dehydrogenase, dependent upon oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate and distinct from that induced by gamma-aminobutyrate, was gratuitously induced by succinic semialdehyde.     

Involvement of the protocatechuate pathway in the metabolism of mandelic acid by Aspergillus niger

Cell-free extracts of Aspergillus niger UBC 814 grown in the presence of dl-mandelate oxidized both d(-)- and l(+)-mandelate via benzoylformate and benzaldehyde to benzoate. dl-p-Hydroxymandelate was oxidized, presumably through a parallel pathway, to p-hydroxybenzoate. A particulate d(-)-mandelate dehydrogenase and a supernatant fraction l(+)-mandelate dehydrogenase converted their respective substrates to benzoylformate. Both flavine adenine dinucleotide and flavine mononucleotide showed a stimulatory effect on the activity of the l(+)-mandelate dehydrogenase. Benzoylformate was decarboxylated to benzaldehyde by an enzyme requiring thiamine pyrophosphate for maximal activity. Two benzaldehyde dehydrogenases dependent on nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, for their activity dehydrogenated benzaldehyde to benzoate. In the presence of reduced NADP (NADPH), benzoate was oxidized via p-hydroxybenzoate and protocatechuate. Reduced NAD could not replace NADPH. Sensitive methods of assay for d(-)-mandelate dehydrogenase and benzoylformate decarboxylase are described. The fungal pathway is compared with these systems in bacteria.