Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of ß-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 µM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 µM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

L-Xylo-3-hexulose, a new rare sugar produced by the action of acetic acid bacteria on galactitol, an exception to Bertrand Hudson's rule.

BACKGROUND: In acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases. METHODS: Membrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor. RESULTS: In this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose. CONCLUSIONS: This reaction may represent an exception to Bertrand Hudson's rule. GENERAL SIGNIFICANCE: Bertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory.

Fungal PQQ-dependent dehydrogenases and their potential in biocatalysis.

In 2014, the first fungal pyrroloquinoline-quinone (PQQ)-dependent enzyme was discovered as a pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH). This discovery laid the foundation for a new Auxiliary Activities (AA) family, AA12, in the Carbohydrate-Active enZymes (CAZy) database and revealed a novel enzymatic activity potentially involved in biomass conversion. This review summarizes recent progress made in research on this fungal oxidoreductase and related enzymes. CcPDH consists of the catalytic PQQ-binding AA12 domain, an N-terminal cytochrome b AA8 domain, and a C-terminal family 1 carbohydrate-binding module (CBM1). CcPDH oxidizes 2-keto-d-glucose (d-glucosone), l-fucose, and rare sugars such as d-arabinose and l-galactose, and can activate lytic polysaccharide monooxygenases (LPMOs). Bioinformatic studies suggest a widespread occurrence of quinoproteins in eukaryotes as well as prokaryotes.

Study of Aberrant Modifications in Peptides as a Test Bench to Investigate the Immunological Response to Non-Enzymatic Glycation.

A side effect of diabetes is formation of glycated proteins and, from them, production of advanced early glycation end products that could determine aberrant immune responses at the systemic level. We investigated a relevant aberrant post-translational modification (PTM) in diabetes based on synthetic peptides modified on the lysine side chain residues with 1-deoxyfructopyranosyl moiety as a possible modification related to glycation. The PTM peptides were used as molecular probes for detection of possible specific autoantibodies developed by diabetic patients. The PDC-E2(167-186) sequence from the pyruvate dehydrogenase complex was selected and tested as a candidate peptide for antibody detection. The structure-based designed type I' ß-turn CSF114 peptide was also used as a synthetic scaffold. Twenty-seven consecutive type 1 diabetic patients and 29 healthy controls were recruited for the study. In principle, the 'chemical reverse approach', based on the use of patient sera to screen the synthetic modified peptides, leads to the identification of specific probes able to characterize highly specific autoantibodies as disease biomarkers of autoimmune disorders. Quite surprisingly, both peptides modified with the (1-deoxyfructosyl)-lysine did not lead to significant results. Both IgG and IgM differences between the two populations were not significant. These data can be rationalized considering that i) IgGs in diabetic subjects exhibit a high degree of glycation, leading to decreased functionality; ii) IgGs in diabetic subjects exhibit a privileged response vs proteins containing advanced glycation products (e.g., methylglyoxal, glyoxal, glucosone, hydroimidazolone, dihydroxyimidazolidine) and only a minor one with respect to (1-deoxyfructosyl)-lysine.

X-ray structure of Arthrobacter globiformis M30 ketose 3-epimerase for the production of D-allulose from D-fructose.

The X-ray structure of ketose 3-epimerase from Arthrobacter globiformis M30, which was previously reported to be a D-allulose 3-epimerase (AgD-AE), was determined at 1.96 Šresolution. The crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 103.98, c = 256.53 Å. The structure was solved by molecular replacement using the structure of Mesorhizobium loti L-ribulose 3-epimerase (MlL-RE), which has 41% sequence identity, as a search model. A hexagonal crystal contained two molecules in the asymmetric unit, and AgD-AE formed a homotetramer with twofold symmetry. The overall structure of AgD-AE was more similar to that of MlL-RE than to the known structures of D-psicose (alternative name D-allulose) 3-epimerases (D-PEs or D-AEs), although AgD-AE and MlL-RE have different substrate specificities. Both AgD-AE and MlL-RE have long helices in the C-terminal region that would contribute to the stability of the homotetramer. AgD-AE showed higher enzymatic activity for L-ribulose than D-allulose; however, AgD-AE is stable and is a unique useful enzyme for the production of D-allulose from D-fructose.

Aromatic aldehydes as selective fluorogenic derivatizing agents for α-dicarbonyl compounds. Application to HPLC analysis of some advanced glycation end products and oxidative stress biomarkers in human serum.

α-Dicarbonyl compounds (α-DCs) are very clinically important as they are considered as advanced glycation end products (AGEs) precursors and biomarkers for many chronic diseases such as diabetes and vascular diseases, in addition to their major role in progression of complications of such diseases. Aromatic aldehydes and ammonium acetate were productively used as a one-pot co-reagents for fluorogenic derivatization of α-DCs yielding fluorescent imidazole derivatives. Among the tried aromatic aldehydes, 4-carbomethoxybenzaldehyde yielded the products with best fluorescent characters. This approach for fluorogenic derivatization of α-DCs overcome the selectivity problem of the most commonly used derivatization reagent for α-DCs, α-diamino compounds, that can react unselectively with α-DCs and aldehydes. Separation of the formed imidazole derivatives of five α-DCs including glucosone, 3-deoxyglucosone, glyoxal, methyl glyoxal and dimethyl glyoxal together with ethylmethylglyoxal as an internal standard was carried out on an octyl column using a mobile phase consisted of methanol-water (15:85, v/v%) containing 0.2% formic acid with time programed flow, followed by fluorescence detection at excitation/emission wavelengths of 310/410 nm. The method showed excellent sensitivity for the targeted α-DCs with limits of detections ranging from 0.4 to 5.0 nM in human serum. Simple protein precipitation procedure was used for human serum treatment yielding very good recovery (91-105%) for the targeted α-DCs. The developed method was fully validated, then applied to the analysis of the five above mentioned clinically important α-DCs in serum samples of healthy, diabetic, rheumatic and cardiac disorders human volunteers. Due to the excellent analytical features of the developed method, including high selectivity and sensitivity, it was able to detect the pattern of the targeted α-DCs serum levels under the investigated different clinical conditions.

Food-derived 1,2-dicarbonyl compounds and their role in diseases.

Reactive 1,2-dicarbonyl compounds (DCs) are generated from carbohydrates during food processing and storage and under physiological conditions. In the recent decades, much knowledge has been gained concerning the chemical formation pathways and the role of DCs in food and physiological systems. DCs are formed mainly by dehydration and redox reactions and have a strong impact on the palatability of food, because they participate in aroma and color formation. However, they are precursors of advanced glycation end products (AGEs), and cytotoxic effects of several DCs have been reported. The most abundant DCs in food are 3-deoxyglucosone, 3-deoxygalactosone, and glucosone, predominating over methylglyoxal, glyoxal, and 3,4-dideoxyglucosone-3-ene. The availability for absorption of individual DCs is influenced by the release from the food matrix during digestion and by their reactivity towards constituents of intestinal fluids. Some recent works suggest formation of DCs from dietary sugars after their absorption, and others indicate that certain food constituents may scavenge endogenously formed DCs. First works on the interplay between dietary DCs and diseases reveal an ambiguous role of the compounds. Cancer-promoting but also anticancer effects were ascribed to methylglyoxal. Further work is still needed to elucidate the reactions of DCs during intestinal digestion and pathophysiological effects of dietary DCs at doses taken up with food and in "real" food matrices in disease states such as diabetes, uremia, and cancer.

When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions.

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3'-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase ß (Polß) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide ("long-patch") BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polß cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polß and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polß-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5'-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polß-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15-30min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polß-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60-70% of the trapped ubiquitin depends on Polß. The mechanism of ubiquitin targeting to Polß-DPC, the subsequent processing of the expected 5'-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.

Facile Enzymatic Synthesis of Ketoses.

Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.

When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.

Near-Infrared Fluorescence Probe for Monitoring the Metabolic Products of Vitamin C in HepG2 Cells under Normoxia and Hypoxia.

Vitamin C (ascorbic acid; AA) is a well-known reducing agent and has been evaluated for its antitumor activity. However, the mechanism for its antitumor action remains unclear. Tracking the metabolism of AA may help to elucidate its antitumor mechanism. In this study, a near-infrared fluorescent probe (Arg-Cy) for monitoring the metabolic products of AA in living cells was developed based on the reaction of the guanidine group in Arg-Cy with the adjacent diketone involved in the metabolites of AA. Consequently, the probe can respond to L-xylosone, a metabolite of AA, with high selectivity and sensitivity and was successfully used to visualize the real-time changes of L-xylosone levels in living cells incubated under normoxic conditions. Considering that the tumor microenvironment suffers from hypoxia, the L-xylosone levels in the process of HepG2 cell death induced by pharmacological doses of AA were also monitored under hypoxic conditions. Surprisingly, no obvious fluorescence change appeared during this process. Furthermore, detection of the intracellular redox state using a reported H2O2 probe confirmed that AA can be metabolized to L-xylosone only under normoxic conditions due to the oxidative stress, but not under hypoxic conditions. Therefore, we hypothesize that the mechanism for cell death induced by AA under hypoxia is different from that under normoxia. Thus, the developed probe can provide a tool for monitoring the metabolism of AA and may help to clarify the mechanism for the antitumor activity of vitamin C in the tumor microenvironment.

Dietary influence on urinary excretion of 3-deoxyglucosone and its metabolite 3-deoxyfructose.

3-Deoxyglucosone (3-DG), a reactive 1,2-dicarbonyl compound derived from d-glucose in food and in vivo, is an important precursor for advanced glycation endproducts (AGEs). At present, virtually no information about the metabolic transit of dietary 3-DG is available. One possible metabolic pathway of 3-DG during digestion is enzymatic transformation to less reactive compounds such as 3-deoxyfructose (3-DF). To study the handling of dietary 1,2-dicarbonyl compounds by the human body, 24 h urinary excretion of 3-DG and its metabolite, 3-deoxyfructose, was investigated. Urinary 3-DG and 3-DF excretion was monitored for nine healthy volunteers following either a diet with no dietary restrictions or a diet avoiding the ingestion of 3-DG and other Maillard reaction products ("raw food" diet). During the "raw food" diet, the urinary 3-DG and 3-DF excretion decreased approximately to 50% compared to the excretions during the diet with no restrictions. When subjects received a single dose of wild honey (50 g) naturally containing a defined amount of 3-DG (505 µmol), median excretion of 3-DG and 3-DF increased significantly from 4.6 and 77 to 7.5 and 147 µmol/day, respectively. The obtained experimental data for the first time demonstrate a dietary influence on urinary 3-DG and 3-DF levels in healthy human subjects.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of a putative xylose isomerase from Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron BT0793, a putative xylose isomerase, was overexpressed in Escherichia coli, purified and crystallized using polyethylene glycol monomethyl ether 550 as the precipitant. X-ray diffraction data were collected to 2.10 Šresolution at 100 K using synchrotron X-rays. The crystal was found to belong to space group P1, with unit-cell parameters a=96.3, b=101.7, c=108.3 Å, α=82.8, ß=68.2, γ=83.0°. The asymmetric unit contained eight subunits of xylose isomerase with a crystal volume per protein weight (VM) of 2.38 Å3 Da(-1) and a solvent content of 48.3%.

L-xylo-3-hexulose reductase is the missing link in the oxidoreductive pathway for D-galactose catabolism in filamentous fungi.

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line.

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,ß-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

Two new tryptamine derivatives, leptoclinidamide and (-)-leptoclinidamine B, from an Indonesian ascidian Leptoclinides dubius.

Two new tryptamine-derived alkaloids, named as leptoclinidamide (1) and (-)-leptoclinidamine B (2), were isolated from an Indonesian ascidian Leptoclinides dubius together with C²-α-D-mannosylpyranosyl-L-tryptophan (3). The structure of 1 was assigned on the basis of spectroscopic data for 1 and its N-acetyl derivative (4). Compound 1 was an amide of tryptamine with two ß-alanine units. Although the planar structure of 2 is identical to that of the known compound (+)-leptoclinidamine B (5), compound 2 was determined to be the enantiomer of 5 based on amino acid analysis using HPLC methods. Compounds 1 to 4 were evaluated for cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma) cells, but none of the compounds showed activity.

Identification of the galactitol dehydrogenase, LadB, that is part of the oxido-reductive D-galactose catabolic pathway in Aspergillus niger.

For the catabolism of D-galactose three different metabolic pathways have been described in filamentous fungi. Apart from the Leloir pathway and the oxidative pathway, there is an alternative oxido-reductive pathway. This oxido-reductive pathway has similarities to the metabolic pathway of L-arabinose, and in Trichoderma reesei (Hypocrea jecorina) and Aspergillus nidulans the same enzyme is employed for the oxidation of L-arabitol and galactitol. Here we show evidence that in Aspergillus niger L-arabitol dehydrogenase (LadA) is not involved in the D-galactose metabolism; instead another dehydrogenase encoding gene, ladB, is induced in response to D-galactose and galactitol and functions as a galactitol dehydrogenase. Deletion of ladB in A. niger results in growth arrest on galactitol and significantly slower growth on D-galactose supplemented with a small amount of D-xylose. D-galactose alone cannot be utilised by A. niger and the addition of D-xylose stimulates growth on D-galactose via transcriptional activation of the D-xylose-inducible reductase gene, xyrA. XyrA catalyses the first step of the D-galactose oxido-reductive pathway, the reduction to galactitol, which in turn seems to be an inducer of the downstream genes such as LadB. The deletion of xyrA results in reduced growth on D-galactose. The ladB gene was expressed in the heterologous host Saccharomyces cerevisiae and the tagged and purified enzyme characterised. LadB and LadA have similar in vitro activity with galactitol. It was confirmed that the reaction product of the LadB reaction from galactitol is L-xylo-3-hexulose as in the case of the T. reesei Lad1.

Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria.

In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes.

Bioconversion of D-glucose into D-glucosone by glucose 2-oxidase from Coriolus versicolor at moderate pressures.

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of D-glucose at carbon 2 in the presence of molecular O2 producing D-glucosone (2-keto-glucose and D-arabino-2-hexosulose) and H2O2. It was used to convert D-glucose into D-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of D-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H2O2 acted as inhibitor for this reaction. The rate of bioconversion of D-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO2 at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55 °C) and pH (5.0) of D-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E (a)) was 32.08 kJ mol⁻¹ and kinetic parameters (V(max), K(m), K(cat) and K(cat)/K(m)) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of D-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.

1,5-anhydro-D-fructose and its derivatives: biosynthesis, preparation and potential medical applications.

1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with α-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.