Dehydrogenase in carbohydrate metabolism in larvae of the silkworm, Bombyx mori L.
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Animals↗
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Enzyme systems in Tetrahymena geleii S. I. anaerobic dehydrogenases concerned with carbohydrate oxidation.
The rates of activity of the dehydrogenase systems in Tetrahymena, which are concerned with carbohydrate oxidation, in descending order of activity are: lactic > isocitric > succinic = glucose > glucose-6-phosphate = 6-phosphogluconic = malic > glutamic = cytochrome linked alpha-glycerophosphate dehydrogenase. No evidence was obtained to indicate the presence of DPN linked alpha-glycerophosphate dehydrogenase.
Carbohydrates↗
Structure of 3-O-(alpha-D-glucopyranosyluronic acid)-L-galactopyranose, an aldobiouronic acid isolated from the polysaccharides of various unicellular red algae.
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Carbohydrate Conformation↗
Atlantic hagfish cardiac muscle: metabolic basis of tolerance to anoxia.
Oxygen tensions in the major venous inputs to the systemic and portal-vein hearts of normoxic Atlantic hagfish (12.3 +/- 1.7 and 11.0 +/- 1.6 mmHg, respectively) are low compared with typical vertebrate values. Anoxia and poisoning with cyanide and azide do not significantly affect in situ performance of the systemic heart. Idoacetate poisoning, however, results in a significant decrease in cardiac performance of the systemic heart to 12% of the initial value after 3 h. Activities of mitochondrial enzymes of hagfish ventricle suggest a small potential for aerobic metabolism compared with those in the aerobic ventricle of Atlantic cod. Activities of enzymes of carbohydrate metabolism indicate similar anaerobic capacity in hagfish and cod ventricle. The ratio of pyruvate kinase to cytochrome c oxidase, an index of anaerobic to aerobic capacity, is 5.6 times greater in hagfish than cod ventricle. Metabolite concentrations in freeze-clamped ventricles of normoxic and hypoxic hagfish indicate hypoxia-induced activation of glycogenolysis, enhanced substrate flow across 6-phosphofructokinase, and an apparent secondary constriction of glycolysis at the level of glyceraldehyde-phosphate dehydrogenase. Carbohydrate utilization via the glycolytic pathway appears essential for maintenance of cardiac performance in both normoxic and anoxic hagfish. Under conditions of severe hypoxia, ATP provision is probably met by anaerobic glycolysis.
Alcohol Oxidoreductases↗
Effects of adenosine on glucuronidation and uridine diphosphate glucuronic acid (UDPGA) synthesis in isolated rat hepatocytes.
Dibutyryl cyclic adenosine 3':5'-monophosphate (DBcAMP) has been shown to inhibit glucuronidation of p-nitrophenol in a concentration-dependent manner in isolated rat hepatocytes. Adenosine (ADO) also decreased glucuronidation in a similar fashion. The effects of adenosine were examined on the variables controlling glucuronidation in intact cells. The addition of adenosine was without effect on either glucuronyltransferase or beta-glucuronidase. Adenosine decreased uridine diphosphate glucuronic acid (UDPGA) levels by 62% and, subsequently, inhibited glucuronidation by 41% in isolated rat hepatocytes. Since the synthesis of UDPGA requires NAD+ for the dehydrogenation of UDP-glucose, alterations in the redox state could account for the decrease in intracellular UDPGA levels. The effects of ADO (500 microM) on lactate and pyruvate content and redox state were examined in rat hepatocytes. ADO caused a 2.1-fold increase in lactate levels and a 2.65-fold increase in the [lactate]/[pyruvate] ratio. The NAD+/NADP ratio, therefore, was decreased by 63% in the presence of ADO. Carbohydrate reserve also affects UDPGA levels; thus, graded concentrations of glucose (5.5, 25, and 50 mM) were added to cells incubated with ADO. At 5.5 mM glucose, ADO caused a 61% decrease in glucuronide formation, while at concentrations of 25 and 50 mM glucose, the inhibition was diminished by 53 and 47% respectively. ADO appears to have decreased the synthesis of UDPGA by decreasing the NAD+/NADH ratio, thus inhibiting UDP-glucose dehydrogenase. Carbohydrate reserve also appears to be involved in the inhibition of glucuronidation mediated by ADO.
Adenine Nucleotides↗
Hereditary hemolytic anemia due to enzyme defects of glycolysis.
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Adenosine Diphosphate↗
A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.
The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.
Aggregatibacter actinomycetemcomitans↗
UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.
The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.
3-Deoxy-7-Phosphoheptulonate Synthase↗
Exopolysaccharide alginate synthesis in Pseudomonas aeruginosa: enzymology and regulation of gene expression.
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Alginates↗
An enzyme module system for the synthesis of dTDP-activated deoxysugars from dTMP and sucrose.
A flexible enzyme module system is presented that allows preparative access to important dTDP-activated deoxyhexoses from dTMP and sucrose. The strategic combination of the recombinant enzymes dTMP-kinase and sucrose synthase (SuSy), and the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-beta-L-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, +/-RmlB) yielded the precursor dTDP-alpha-D-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-alpha-D-glucose (3) on a 0.2-0.6 g scale with overall yields of 62 % and 72 %, respectively. A two-step strategy in which the SuSy module was followed by the deoxysugar module (RmlC and RmlD) resulted in the synthesis of dTDP-beta-L-rhamnose (4; 24.1 micromol, overall yield: 35.9 %). Substitution of RmlC by DnmU from the dTDP-beta-L-daunosamine pathway of Streptomyces peucetius in this module demonstrated that DnmU acts in vitro as a 3,5-epimerase with 3 as substrate to yield 4 (32.2 mumol, overall yield: 44.7 %). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-alpha-D-quinovose (6) and dTDP-alpha-D-fucose (7) in a ratio of 2:1. In summary, the modular character of the presented enzyme system provides valuable compounds for the biochemical characterization of deoxysugar pathways playing a major role in microbial producers of antibiotic and antitumour agents.
Base Sequence↗
Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry.
Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of pyranose oxidases is D-glucose which is converted to 2-keto-D-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-D-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-D-glucose can serve as an intermediate in an industrial process for the conversion of D-glucose into D-fructose has stimulated research on the use of pyranose oxidases in biotechnical applications. Unique catalytic potentials of pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs.
Basidiomycota↗
(Chemo)enzymatic synthesis of dTDP-activated 2,6-dideoxysugars as building blocks of polyketide antibiotics.
The flexible substrate spectrum of the recombinant enzymes from the biosynthetic pathway of dTDP-beta-L-rhamnose in Salmonella enterica, serovar typhimurium (LT2), was exploited for the chemoenzymatic synthesis of deoxythymidine diphosphate- (dTDP-) activated 2,6-dideoxyhexoses. The enzymatic synthesis strategy yielded dTDP-2-deoxy-alpha-D-glucose and dTDP-2,6-dideoxy-4-keto-alpha-D-glucose (13) in a 40-60 mg scale. The nucleotide deoxysugar 13 was further used for the enzymatic synthesis of dTDP-2,6-dideoxy-beta-L-arabino-hexose (dTDP-beta-L-olivose) (15) in a 30-mg scale. The chemical reduction of 13 gave dTDP-2,6-dideoxy-alpha-D-arabino-hexose (dTDP-alpha-D-olivose) (1) as the main isomer after product isolation in a 10-mg scale. With 13 as an important key intermediate, the in vitro characterization of enzymes involved in the biosynthesis of dTDP-activated 2,6-dideoxy-, 2,3,6-trideoxy-D- and L-hexoses can now be addressed. Most importantly, compounds 1 and 15 are donor substrates for the in vitro characterization of glycosyltransferases involved in the biosynthesis of polyketides and other antibiotic/antitumor drugs. Their synthetic access may contribute to the evaluation of the glycosylation potential of bacterial glycosyltransferases to generate hybrid antibiotics.
Animals↗
Screening of sugar converting enzymes using quantitative MALDI-ToF mass spectrometry.
Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes.
Carbohydrate Dehydrogenases↗
Isolation and characterization of the rml gene homologs from Porphyromonas gingivalis.
We cloned four genes from the Porphyromonas gingivalis chromosome, the gene products of which catalyze the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate when they were expressed in Escherichia coli. The amino acid sequences deduced from these genes showed significant homology to proteins encoded by the rml genes involved in dTDP-L-rhamnose biosynthesis in other gram-negative bacteria. Reverse transcriptase polymerase chain reaction analysis revealed that these four genes are expressed as a single transcript in P. gingivalis. To clarify the role of the rml gene homologs in this organism, construction of mutants defective in the rml gene homologs was attempted by allelic exchange. Unexpectedly, any mutants defective in the rml gene homologs were unable to be isolated, and the allelic exchange was possible only if the wild-type rml gene homologs were present on the chromosome. These results suggest that the rml gene homologs might be essential for the viability of P. gingivalis under the culture conditions used in this study.
Bacterial Proteins↗
Some properties of cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum.
Cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum has been purified to homogeneity by a new procedure. The carbohydrate and amino acid compositions of the enzyme have been determined. Cellobiose oxidase contains FAD and cytochrome b prosthetic groups. Mr of the enzyme has been estimated at 74400 by sedimentation equilibrium. The enzyme is a monomer. Optical, fluorescence and e.p.r. spectra of oxidized and reduced cellobiose oxidase have been determined. A preliminary investigation of the substrate specificity of cellobiose oxidase reveals that disaccharides and even some insoluble polysaccharides are substrates, but not monosaccharides. Strong substrate inhibition is seen at high concentrations of cellobiose. This effect is particularly marked when oxygen is the electron acceptor. Cellobiose oxidase is unusual among flavoproteins, since it stabilizes the red anionic flavin semiquinone and forms a sulphite adduct, yet appears to produce the superoxide anion as its primary reduced oxygen product.
Amino Acids↗
A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale.
A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (< or = 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O2 or other acceptors. When D-glucose served as electron-donating substrate, an activity of 2 s(-1) was observed at pH 5.5 and 23 degrees C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.
Alcohol Oxidoreductases↗
Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms.
The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.
Alginates↗
Characterization of dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP-4-dehydrorhamnose reductase, required for dTDP-L-rhamnose biosynthesis in Salmonella enterica serovar Typhimurium LT2.
The thymidine diphosphate-L-rhamnose biosynthesis pathway is required for assembly of surface glycoconjugates in a growing list of bacterial pathogens, making this pathway a potential therapeutic target. However, the terminal reactions have not been characterized. To complete assignment of the reactions, the four enzymes (RmlABCD) that constitute the pathway in Salmonella enterica serovar Typhimurium LT2 were overexpressed. The purified RmlC and D enzymes together catalyze the terminal two steps involving NAD(P)H-dependent formation of dTDP-L-rhamnose from dTDP-6-deoxy-D-xylo-4-hexulose. RmlC was assigned as the thymidine diphosphate-4-dehydrorhamnose 3,5-epimerase by showing its activity to be NAD(P)H-independent. Spectrofluorometric and radiolabeling experiments were used to demonstrate the ability of RmlC to catalyze the formation of dTDP-6-deoxy-L-lyxo-4-hexulose from dTDP-6-deoxy-D-xylo-4-hexulose. Under reaction conditions, RmlC converted approximately 3% of its substrate to product. RmlD was unequivocally identified as the thymidine diphosphate-4-dehydrorhamnose reductase. The reductase property of RmlD was shown by equilibrium analysis and its ability to enable efficient biosynthesis of dTDP-L-rhamnose, even in the presence of low amounts of dTDP-6-deoxy-L-lyxo-4-hexulose. Comparison of 23 known and predicted RmlD sequences identified several conserved amino acid residues, especially the serine-tyrosine-lysine catalytic triad, characteristic for members of the reductase/epimerase/dehydrogenase protein superfamily. In conclusion, RmlD is a novel member of this protein superfamily.
Amino Acid Sequence↗