Mannitol Dehydrogenase Research Articles

Regulation of mannitol catabolism in Candida albicans: evidence for cyclic AMP-independent glucose effect.

Candida albicans was examined for a glucose effect and showed typical diauxic growth on a mixture of glucose and mannitol, in which mannitol utilization occurred only after exhaustion of glucose. The activity of NAD-linked mannitol dehydrogenase was very low while glucose was present in the medium, but started to increase after consumption of glucose. This increase in activity was fully prevented by trichodermin, an inhibitor of protein synthesis. The uptake of mannitol was detected in the cells grown on mannitol, but not in those grown on glucose with or without mannitol. Mannitol uptake by mannitol-grown cells was not affected by the presence of glucose (0.2 g l-1). These findings indicate that in C. albicans glucose represses the inducible syntheses of mannitol dehydrogenase and a mannitol transport system, and that the involvement of inducer exclusion in this effect is unlikely. Fructose, and to lesser extents galactose, mannose and sucrose, also exhibited similar effects on mannitol metabolism. No correlation was found between the intracellular cyclic AMP levels and the glucose effect.

The Agaricus bisporus mannitol pathway during sporophore growth

Fructose 6-phosphatase and fructokinase activities were identified in Agaricus bisporus fruit body extracts. These enzymes and mannitol dehydrogenase (MDH; E.C. 1.1.1.138) were assayed during the growth of fruit bodies on compost. MDH activity was high at early stages of development, and declined during growth to very low levels after sporulation. Phosphatase activity rose to a maximum during late fruit body development, and then fell to a low level after sporulation. Fructokinase activity was present throughout development and maximum activity was observed at late development. The results are discussed in relation to previously observed changes in fruit body mannitol content during development.

Mannitol Metabolism in Agaricus bisporus: Purification and Properties of Mannitol Dehydrogenase

Summary: Mannitol dehydrogenase (EC 1.1.1.138) has been purified to homogeneity from fruit bodies of Agaricus bisporus. M r values of 115000 were determined by gel filtration and 130000 by rate zonal ultracentrifugation. The sedimentation coefficient is 6.5S. The native protein is composed of four subunits of M r 29000. The enzyme is specific for NADP, and shows low activity with sorbitol. Normal Michaelis-Menten kinetics are exhibited for both mannitol and NADP, giving K m values of 16.2 mm and 36μm respectively at pH 7.0. The K m value for NADPH is 38.5μm and that for fructose approximately 1.2 m. The V max is 591 μm min−1 (mg protein)−1 for mannitol synthesis and 5μmol min−1 (mg protein)−1 for fructose synthesis at pH 7.0. Inhibition of fructose synthesis by NADPH is stronger than inhibition of mannitol synthesis by NADP. The results are discussed with respect to the control of enzyme activity under physiological conditions.

Maintenance of Different Mannitol Uptake Systems during Starvation in Oxidative and Fermentative Marine Bacteria

The mannitol uptake systems in marine Vibrio and Pseudomonas isolates from the kelp beds off the South African west coast were examined. The fermentative Vibrio isolate possessed a constitutive rapid mannitol uptake system and also a soluble mannitol-1-phosphate dehydrogenase, indicative of a mannitol phosphotransferase system. An inducible, relatively less active mannitol uptake system was detected in the oxidative Pseudomonas isolate, and this strain possessed a mannitol dehydrogenase. The maintenance of these systems during starvation survival was studied. The Vibrio isolate maintained its initial uptake system for approximately 5 weeks of starvation, after which time the uptake system was replaced by one with a higher affinity for mannitol. The mannitol transport system of the Pseudomonas isolate was depressed early in starvation (30 h) but could be readily induced by exogenous mannitol after 6 weeks of starvation. The relative proportions of mannitol which was incorporated and respired were determined in starved Vibrio and Pseudomonas strains.

Strain identification in Rhizobium by starch gel electrophoresis of isoenzymes

Sonicated extracts of rhizobia, especiallyRhizobium leguminosarum from pea and vetch, were run in horizontal starch gel electrophoresis in the cold. The rhizobia were grown on agar on a slime suppressing substrate of tryptone-yeast extract-CaCl2 with small amounts of mannitol, sorbitol and arabinose and other sugars as enzyme inducers. After electrophoresis the gels were separated into several slabs by a gel cutter. Each slab was stained for a particular enzyme. Among numerous enzyme systems tested we found useful variation in esterases (EC 3.1.1.1., EC 3.1.1.2), 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), mannitol dehydrogenase (EC 1.1.1.67), and arabinose dehydrogenase (EC 1.1.1.46). It was possible to distinguish at least 7 different types of pea rhizobia among 16 strains isolated from one batch of 5 kg soil.

Polyol Metabolism in Fungi

Polyols (sugar alcohols) are widely distributed in fungi. They could serve several roles in living organisms: (1)act as carbohydrate reserves, (2) act as translocatory compounds, (3) function in osmoregulation, and (4) in coenzyme regulation and storage of reducing power. This chapter describes several enzymes that are concerned with polyol metabolism in fungi. These include Glycerol dehydrogenase (NAD + ), Glycerol-3-phosphate dehydrogenase (NAD + ), Xylitol dehydrogenase (NAD + ), Mannitol-1-phosphate dehydrogenase, Polyol dehydrogenase (NADP + ), Mannitol dehydrogenase (NAD + ), Glyceroldehydrogenase (NADP + ), Ribitol-5-phosphate dehydrogenase (NADP + ), Mannitold ehydrogenase(NADP + ), Glycerophosphate dehydrogenase, Glycerol kinase, Mannitol kinase, Mannitol-1-phosphatase, Sorbitol (glucitol) dehydrogenase, and Glycerol oxidase. A number of enzymes have relatively high K m , values for their substrates and a high pH optimum for the oxidation of polyols, which is likely to be a consequence of the buffer more effectively consuming protons at such pH values. It is conceivable that these particular enzymes are membrane-bound. The groups to which the various fungi belong are identified and designated as—namely, Mastigomycotina (M), Zygomycotina (Z), Ascomycotina (A), Deuteromycotina (D) and Basidiomycotina (B).

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.

Caffeine inhibition of aflatoxin synthesis: probable site of action.

Aflatoxin production by pregrown cultures of Aspergillus parasiticus was completely inhibited by incorporation of 2 mg of caffeine per ml into the medium. This was accompanied by a decrease in glucose utilization and an inhibition of oxygen uptake and carbon dioxide evolution. Enzyme analyses indicated no significant differences in specific activities on glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphofructokinase, fructose 1,6-diphosphatase, pyruvate kinase, or malate dehydrogenase. Glucose uptake kinetics indicated a linear dose-related inhibition of glucose uptake. It appears likely that caffeine inhibits aflatoxin synthesis by restricting the uptake of carbohydrates which are ultimately used by the mold to synthesize this family of mycotoxins.

Mannitol dehydrogenase from industrial waste mycelium of Penicillium chrysogenum. Purification, properties and possible use in cofactor regenerating systems in vitro

Mannitol dehydrogenase, NADP specific (EC 1.1.1.138), was purified 823 fold from industrial waste mycelium of Penicillium chrysogenum. The enzyme had a molecular weight of 160,000 and was composed of four subunits of equal size. The pH optimum for activity was 7.3 and for stability 6.0.

Purification and characterization of mannitol dehydrogenase from Aspergillus parasiticus.

Mannitol dehydrogenase, NADP specific (EC 1.1.1.138), was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.4 X 10(5) and was composed of four subunits of apparently equal size. The substrate specificity was limited to D-mannitol, D-glucitol, D-arabinitol, 1-deoxy-D-mannitol, and 1-deoxy-D-glucitol. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to mannitol, with Ki and 1 microM. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of mannitol dehydrogenase.

Characterization of an inducible mannitol-binding protein fromPseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO formed a mannitol-binding protein that was coinducible with mannitol uptake activity. One species of mannitol-binding protein was isolated from disrupted cell suspensions and the protein also was released from mannitol-grown cells by osmotic shock treatment. The mannitol-binding protein )molecular weight ∼37,000) was purified 49-fold and resolved from the enzyme mannitol dehydrogenase (molecular weight ∼85,000), which did not exhibit mannitol-binding activity. Mannitol-binding protein had an isoelectric point (pI) of 8.3 and an apparent dissociation constant (K D ) of 2.3 μM for mannitol. Mannitol binding was unaffected by glucose and glycerol in competitive inhibition studies, although mannose, fructose, and sorbitol caused 5 to 20% inhibition of binding at concentrations equimolar with mannitol.

Enzymes related to fructose utilization in Pseudomonas cepacia.

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.

VARIATIONS IN ENZYME ACTIVITY DURING PERIODIC FRUITING OF AGARICUS BISPORUS

SummaryGlucose‐6‐phosphate dehydrogenase (G6PD), glucosephosphate isomerase (GPI) and mannitol dehydrogenase (MD) were assayed in sporophores harvested at stages 1 (pin) and 2 (button) of development throughout several fruiting cycles. Higher G6PD specific activity was consistently found in stage 1 and 2 sporophores harvested between periods of maximum stage 2 production (flushes). There was minimum G6PD activity during the flushes. At stage 1 the range of variation was greater and the timing of the peaks in the flush cycle more uniform than in stage 2 sporophores. GPI and MD activities also varied with flushing, but showed a less consistent pattern than G6PD. MD normally exhibited greatest activity in stage 1 and 2 sporophores between flushes. GPI activity was higher between flushes in stage 1 sporophores, and during flushes at stage 2.Mannitol and trehalose were highest in stage 1 sporophores harvested between flushes. The results are discussed in relation to the mechanism of flushing.

Sorbitol metabolism by apple seedlings

The aims of this work were to compare the roles of sorbitol and sucrose in seedlings of Malus domestica, to discover which tissues synthesize sorbitol and which break it down, and to examine these tissues for enzymes of sorbitol metabolism. The detailed distribution of label was determined after supplying intact seedlings with 14CO 2, and excised parts of seedlings with [U- 14C]fructose and [U- 14C]sorbitol. The results showed that appreciable synthesis of sorbitol occurred only in the leaves but did not depend directly on photosynthesis. All tissues examined metabolized sorbitol but metabolism was extensive only in root apices, and in leaves which had been kept in the dark. The above experiments suggest that sorbitol supplements but does not replace sucrose. Extracts of apple leaves showed no trace of either a polyol or a polyol phosphate dehydrogenase but did exhibit sorbitol-6-phosphate phosphatase activity. A limited number of experiments with extracts of the blades of Laminaria digitata indicated that they contained mannitol-1-phosphate phosphatase and mannitol dehydrogenase.

Improved enzymatic method for determining mannitol and its application to dog serum after mannitol infusion.

An enzymatic method for mannitol quantitation has been developed. Its application mannitol levels in serum samples from dogs after mannitol infusion is illustrated. The method is based on the spectrophotometric measurement of the initial rate of NADH formation in the reaction between mannitol and NAD catalyzed by mannitol dehydrogenase. A linear relationship between initial rates and mannitol concentration is seen between 17 mumol/l and 6.7 mmol/l mannitol. From the extent of dilution of the serum sample in the assay this corresponds to serum levels of 0.5 to 200 mmol/l. Because of the high degree of substrate specificity of mannitol dehydrogenase and the extensive dilution of the serum sample in the assay reaction mixture, serum proteins and glucose do not interfere with the reaction. As a result, pre-treatment of samples to remove glucose and deproteinization are necessary.

Properties of Tn5-induced Carbohydrate Mutants in Rhizobium meliloti

SUMMARY: Carbohydrate mutants were isolated from Rhizobium meliloti L5-30 using the translocatable drug-resistance element Tn5. Enzyme assays with cell-free extracts of four mutants showed that they lacked mannitol dehydrogenase, ribose kinase, xylose isomerase and fructose kinase, respectively. An l-arabinose mutant was also isolated. Uptake studies showed that the ribose, xylose and fructose mutants still utilized the sugars on which they were unable to grow, possibly indicating that R. meliloti possesses alternative metabolic routes which do not result in growth. All the mutants were able to nodulate alfalfa plants, but the fructose kinase mutant was unable to fix nitrogen and the l-arabinose mutant showed either late or no nitrogen-fixing ability.

The distribution of the NADPH regenerating mannitol cycle among fungal species.

The mannitol cycle is an important NADPH regenerating system in Alternaria alternata. The cycle is built up to the following enzymes: mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, mannitol dehydrogenase and hexokinase. The net reaction of one cycle turn is: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The enzymes needed for an operating cycle were found in Aspergillus, Botrytis, Penicillium, Pyricularia, Trichothecium, Cladosporium and Thermomyces all genera belonging to Fungi Imperfecti. The only genus of this class lacking the cycle was Candida. No genera from the classes Basidiomycetes and Phycomycetes showed any mannitol 1-phosphate dehydrogenase or mannitol 1-phosphatase activities. The genera investigated, belonging to Ascomycetes, Gibberella, Ceratocystis and Neurospora all lacked mannitol 1-phosphate dehydrogenase. It was concluded that the mannitol cycle is an important and widespread pathway for NADH oxidation and NADP+ reduction in the organisms belonging to the class Fungi Imperfecti.

Some effects of mannitol on the glucose metabolism of two derivatives of a strain ofRhizobium japonicum differing in mannitol dehydrogenase

The effects of mannitol were investigated by comparing some metabolic features in colonial derivatives, I-110 and L1-110, ofRhizobium japonicum strain 3IIb110, grown either on glucose alone (G-cells) or in glucose media supplemented with mannitol (GM-cells). The polyol stimulated the synthesis of not only mannitol dehydrogenase, which is active in derivative L1-110, but also the nicotinamide adenine dinucleotide (NAD)-linked 6-phosphogluconate (6-PG) dehydrogenase (EC 1.1.1.43). As revealed by radiorespirometry, when GM-cells were allowed to metabolize glucose, they produced relatively more CO2 from the first and sixth carbons of the sugar than G-cells did. This finding is evidence that NAD-linked 6-PG dehydrogenase might initiate an unknown pathway different from the hexose cycle and the pentose phosphate (PP) pathway. Mannitol exerted no allosteric control on the oxygen consumption and the glucose transport systems. Active uptake of the polyol was correlated with the presence of mannitol dehydrogenase (EC 1.1.1.67); it did not hinder the transport of glucose even though both systems derive their energy for active transport from a common source presumptively characterized as the energized membrane state. Mannitol, however, suppressed by two- or threefold the glucose uptake system. Addition of the polyol to the cell suspensions of both colonial types ofR. japonicum metabolizing glucose caused an immediate 40–50% drop of adenosine triphosphate (ATP) concentrations, owing in part to the mannitol kinase reaction. Type I-110 failed to overcome this reduction of ATP levels, and low growth rates could results. In contrast, type L1-110 offsets the reduction of ATP concentration by oxidizing mannitol as an additional source of energy through mannitol dehydrogenase, fructokinase, and a sequence of glycolytic reactions. The polyol also induced type L1-110 to produce extracellular slimy materials that, apparently, harbor amounts of ATP and proteins.

Production of NADPH in the mannitol cycle and its relation to polyketide formation in Alternaria alternata.

The enzymes mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.

Mannitol and Fructose Catabolic Pathways of Pseudomonas aeruginosa Carbohydrate-Negative Mutants and Pleiotropic Effects of Certain Enzyme Deficiencies

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.