2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine

D-Glucosamine has been widely reported to have immunosuppressive actions on neutrophils, lymphocytes, and other cells of the immune system. However, under conditions used in biological experiments (e.g., neutral pH, and phosphate buffers), we have found that D-glucosamine self-reacts to form 2,5-deoxyfructosazine [2-(D-arabino-tetrahydroxybutyl)-5-(D-erythro-2,3,4-trihydroxybutyl)pyrazine] (1) and 2,5-fructosazine [2,5-bis(D-arabino-tetrahydroxybutyl)pyrazine] (2). When tested for bioactivity at nontoxic concentrations, these D-glucosamine derivatives were more effective inhibitors of IL-2 release from PHA-activated T cells than d-glucosamine. Hence, fructosazines constitute a novel class of immunomodulators.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

D-Erythroascorbic acid activates cyanide-resistant respiration in Candida albicans

Higher plants, protists and fungi possess cyanide-resistant respiratory pathway, which is mediated by alternative oxidase (AOX). The activity of AOX has been found to be dependent on several regulatory mechanisms including gene expression and posttranslational regulation. In the present study, we report that the presence of cyanide in culture medium remarkably retarded the growth of alo1/alo1 mutant of Candida albicans, which lacks d-arabinono-1,4-lactone oxidase (ALO) that catalyzes the final step of d-erythroascorbic acid (EASC) biosynthesis. Measurement of respiratory activity and Western blot analysis revealed that increase in the intracellular EASC level induces the expression of AOX in C. albicans. AOX could still be induced by antimycin A, a respiratory inhibitor, in the absence of EASC, suggesting that several factors may act in parallel pathways to induce the expression of AOX. Taken together, our results suggest that EASC plays important roles in activation of cyanide-resistant respiration in C. albicans.     

Molecular characterization of D-amino acid oxidase from common carp Cyprinus carpio and its induction with exogenous free D-alanine

A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

Structural basis of D-DOPA oxidation by D-amino acid oxidase: alternative pathway for dopamine biosynthesis

D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathway. Here, we provide direct crystallographic evidence that D-DOPA is readily fitted into the active site of human DAO, where it is oxidized by the enzyme. Moreover, our kinetic data show that the maximal velocity for oxidation of D-DOPA is much greater than for D-serine, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, determination of the structures of human DAO in various states revealed that the conformation of the hydrophobic VAAGL stretch (residues 47-51) to be uniquely stable in the human enzyme, which provides a structural basis for the unique kinetic features of human DAO.     

Ni(II) complexes with Schiff bases derived from amino sugars

It was found by 1H and 13C NMR spectroscopy that the Schiff base, 2-deoxy-2-(2-hydroxybenzaldimino)-D-glucopyranose exhibits enol-imine-keto-amine and anomeric equilibria in methanolic, and in dimethyl sulfoxide solutions. The reaction of the Schiff base with nickel acetate gave the bidentate, mononuclear Ni(II) complex that was characterized by spectroscopic methods and by cyclic voltammetry. The coordination of the Schiff base to the metal is through the enol-imine tautomeric form, and the anomeric equilibrium remains in dimethyl sulfoxide solutions. This complex was also obtained by reaction of D-glucosamine with Ni(II) salicylaldehydate. The same reaction was employed for the synthesis of bis-N-[2-deoxy-D-galactopyranosyl-2-(2-hydroxybenzaldiminate)]Ni(II). The small paramagnetic shifts of the 1H NMR resonances of the complexes suggest that paramagnetic species are present in low proportions.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

Simultaneous measurement of D-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

N-methyl-D-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need D-serine for their channel activities in various brain regions. In mammalian brains, D-serine is produced from L-serine by serine racemase and degraded by D-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, D-serine is also degraded to pyruvate by D-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from D-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4+/-0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7+/-0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

An improved synthesis of 5-thio-D-ribose from D-ribono-1,4-lactone

5-Thio-D-ribopyranose was synthesized from D-ribono-1,4-lactone (1) by two approaches: (i) 5-bromo-5-deoxy-D-ribono-1,4-lactone (2) was successively transformed into 5-bromo-5-deoxy, 5-S-acetyl-5-thio or 5-thiocyanato-D-ribofuranose derivatives; appropriate treatment then lead to 5-thio-D-ribopyranose (7) in 46-48% overall yield and; (ii) 2 was transformed into the 5-S-acetyl-5-thio-D-ribono-1,4-lactone derivative (11). Reduction and deprotection of 11 afforded 5-thio-D-ribopyranose (7) in 57% overall yield.     

An efficient synthesis of methyl 1,3-O-isopropylidene-alpha-D-fructofuranoside and 2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal derivatives from sucrose

Acetalation of sucrose with 2,2-dimethoxypropane in 1,4-dioxane in the presence of p-toluenesulfonic acid, followed by acetylation, afforded methyl 4,6-di-O-acetyl-1,3-O-isopropylidene-alpha-D-fructofuranoside and 4-O-acetyl-2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal as major products, while tosylation of the intermediate acetals provided methyl 6-O-tosyl-1,3-O-isopropylidene-alpha-D-fructofuranose.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

The presence of high concentrations of free D-amino acids in human saliva

Free neutral D-amino acids have previously been detected in human plasma, usually accounting for less than 2% of the total free amino acid concentration (D-amino acid ratio) [Nagata, Y., Masui, R., Akino, T., 1992a. The presence of free D-serine, D-alanine and D-proline in human plasma. Experientia 48, 986-988. Nagata, Y., Yamamoto, K., Shimojo, T., 1992b. Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. Journal of Chromatography 575, 147-152. Nagata, Y., Yamamoto, K., Shimojo, T., Konno, R., Yasumura, Y., Akino, T., 1992c. The presence of free D-alanine, D-proline and D-serine in mice. Biochimca et Biiophysica Acta 1115, 208-211]. In the present study to search for the source of free D-amino acids, D- and L-enantiomers of the major non-essential amino acids, i.e., the free form of serine, alanine, proline, aspartate and glutamate were analyzed by HPLC in human saliva, submandibular glands and oral epithelial cells. The D-enantiomer ratios to total of free alanine or proline were 35% and 20%, respectively, in saliva. The ratios of the other D-amino acids were less than 11%. The effect of ingested food and oral bacteria on the saliva amino acid levels was suggested to be insignificant. D-Alanine and d-aspartate were also detected in the submandibular gland in ratios up to 5%, and D-alanine and d-proline were found in oral epithelial cells in ratios of 18% and 5%, respectively. The submandibular gland and oral epithelial cells are suggested to be possible sources of the saliva D-alanine and D-aspartate.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Esterification of select polyols with D-glucaric acid as model reactions for esterification of starch

Aqueous solutions of D-glucaric acid and model polyols xylitol, methyl alpha-D-glucopyranoside or beta-cyclodextrin were freeze dried, then heated, and the product mixtures analyzed by instrumental methods that included GC-MS, electrospray ionization-mass spectrometry (ESIMS), and NMR. The thermal process and analyses were carried out with these polyols in order to determine to what extent multiple acylations of the alcohol functions occurred with D-glucaric acid lactones serving as acylating agents, the extent to which acylations occurred at the 1 degrees alcohol sites, and the relative tendency for acylations to occur at the C1 or C6 end of the glucaryl unit. The results of these studies showed an overwhelming preference for 1 degrees alcohol acylation and preferred acylation occurring at the C1 end of the glucaryl unit.     

Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.