Deglycosylation of the Isoflavone C-Glucoside Puerarin by a Combination of Two Recombinant Bacterial Enzymes and 3-Oxo-Glucose.

A human intestinal bacterium strain related to Dorea species, PUE, can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose. We reported previously that 3″-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation, and we characterized a bacterial enzyme, the DgpB-DgpC complex, that cleaved the C-glycosidic bond in 3″-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of the C-3″ hydroxyl in puerarin has not been identified. In this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride acceptor. In the redox reaction, NAD(H) functioned as the cofactor, which bound tightly but noncovalently to DgpA. Kinetics analysis of DgpA revealed that the reaction proceeded via a ping-pong mechanism. Enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, the DgpB-DgpC complex, and 3-oxo-glucose. In addition, the metabolite derived from the sugar moiety in the 3″-oxo-puerarin-cleaving reaction catalyzed by the DgpB-DgpC complex was characterized as 1,5-anhydro-d-erythro-hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a ß-elimination-like mechanism.IMPORTANCE One important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more-absorbable aglycones and further degradation products that show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O-deglycosylation have been reported, enzymes responsible for C-deglycosylation are still limited. In this study, we characterized enzymes involved in the C-deglycosylation of puerarin from a human intestinal bacterium, PUE. Here, we report the purification and characterization of a recombinant oxidoreductase involved in C-glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C-deglycosylation.

Expression and Characterization of the Human Intestinal Bacterial Enzyme Which Cleaves the C-Glycosidic Bond in 3″-Oxo-puerarin.

Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA-H). Among them, DgpB-C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.

Enzymatic cleavage of the C-glucosidic bond of puerarin by three proteins, Mn(2+), and oxidized form of nicotinamide adenine dinucleotide.

We previously isolated the human intestinal bacterium, strain PUE, which can cleave the C-glucosidic bond of puerarin to yield its aglycone daidzein and glucose. In this study, we partially purified puerarin C-glucosidic bond cleaving enzyme from the cell-free extract of strain PUE and demonstrated that the reaction was catalyzed by at least three proteins, Mn(2+), and oxidized form of nicotinamide adenine dinucleotide (NAD(+)). We completely purified one of the proteins, called protein C, by chromatographic separation in three steps. The molecular mass of protein C was approximately 40 kDa and the amino acid sequence of its N-terminal region shows high homology to those of two putative proteins which belong to Gfo/Idh/MocA family oxidoreductase. Protein C catalyzed hydrogen-deuterium exchange reaction of puerarin to 2"-deuterated puerarin in D(2)O condition, which closely resembles those of glycoside hydrolase family 4 and 109.

The C-glucosyl bond of puerarin was cleaved hydrolytically by a human intestinal bacterium strain PUE to yield its aglycone daidzein and an intact glucose.

C-Glycosides are usually resistant against acidic hydrolysis and enzymatic treatments because C-1 of the sugar moiety is directly attached to the aglycone by C-C bonding. Nevertheless, a human intestinal bacterium, strain PUE, can cleave the C-glucosyl bond of puerarin to yield its aglycone daidzein. To clarify the mechanism of the cleaving reaction, we tried to identify the structure of the metabolite derived from the sugar moiety of puerarin. To detect it easily, deuterium labeled puerarin, [6″,6″-D(2)]puerarin, was prepared in 7 steps. Sugars contained in a metabolite mixture from [6″,6″-D(2)]puerarin was analyzed by an HPLC-electrospray ionization (ESI)-MS method after treatment of sugars with 1-phenyl-3-methyl-5-pyrazolone (PMP). Since deuterium labeled glucose was detected in the metabolite mixture of [6″,6″-D(2)]puerarin, we concluded that puerarin was metabolized to daidzein and an intact glucose by strain PUE. As C-1 of the sugar was hydroxylated instead of hydrogenating, C-glucosyl bond-cleaving reaction is not reduction but hydrolysis. This is the first report of revealing the reaction manner and the exact products of C-glucosyl bond-cleaving reaction.

New chromane derivatives isolated from the brown alga, Sargassum micracanthum.

Two new chromane type meroterpenoids were isolated from the methanolic extract of the brown alga, Sargassum micracanthum. Their structures were elucidated based on spectroscopic analysis and chemical conversion. The absolute stereochemistry of the methyl group at C-8' in 1 and related compounds were determined by modified Mosher's method.

Effects of plastoquinones from the brown alga Sargassum micracanthum and a new chromene derivative converted from the plastoquinones on acute gastric lesions in rats.

Previously, we reported the anti oxidative and anti viral effects of plastoquinones (compounds 1, 2) extracted from the seaweed Sargassum micracanthum (Kuetzing) Endlicher and a new chromene compound (compound 3), which was converted from the plastoquinones. Recently, we have also demonstrated the antiulcer effects of these compounds and assessed the effects using a rat model of acute gastric lesion and fundus strips isolated from rats. In hydrochloric acid/ethanol rat ulcer tests: 1) oral administrations of compounds 1, 2, and 3 1--10, 3--30 and 10--30 mg/kg, respectively, and omeprazole 3--30 mg/kg showed dose-dependent antiulcer effects: 2) the antiulcer effects after intraduodenal administration of the respective compounds at the dose of 30 mg/kg were found to be significant: and 3) a decrease in the hexosamine level of the gastric mucosa was slightly improved by oral administration of compounds 1, 2, and 3 30 mg/kg. In indomethacin-induced gastric ulcer tests, the antiulcer effects of compounds 1, 2, and 3 10 mg/kg (p.o.) were not significant. Compounds 1, 2, and 3 showed slight contracting effects on the fundus isolated from rats and these effects were inhibited by pretreatment with AH6809, an inhibitor of prostaglandin DP, EP(1), and EP(2) receptors. These results suggest that the protection of the mucosa via endogenous prostaglandins might be related to the antiulcer effects of compounds 1, 2, and 3.

A synthetic study on antiviral and antioxidative chromene derivative.

An efficient synthesis of the antiviral and antioxidative chromene (1) was achieved. A small amount of chromene 1 could be derived from plastoquinones 2 and 3, the major constituents of the brown alga, Sargassum micracanthum. By the following synthetic scheme involving its application, many kinds of analogs can be synthesized for evaluation of their biological activity and mechanistic study. The total synthesis of 1, started from geranyl acetate and protected 2-bromo-6-methylhydroquinone, was executed with Sharpless asymmetric dihydroxylation for introduction of the terminal diol system and base-catalyzed sigmatropic rearrangement for construction of the chromene skeleton as the crucial steps. The stereochemistry at C-11' was reconfirmed by this synthesis.

Solid-phase synthesis and biological activity of a combinatorial cross-conjugated dienone library.

The solid-phase synthesis of a combinatorial cross-conjugated dienone library based on the structure of clavulones and their biological activity are reported. Clavulones are a family of marine prostanoids, and are composed of a cross-conjugated dienone system bearing two alkyl side-chains. The cross-conjugated dienone system irreversibly reacted with two nucleophiles. Our strategy for the solid-phase synthesis of the cross-conjugated dienones involves the Sonogashira-coupling reaction of a solid-supported cyclopentenone 10 bearing an acetylene group, followed by aldol condensation with aldehydes. The diphenyl derivative 7 aA was prepared from the solid-supported cyclopentenone 10 in 56% total yield. Combinatorial synthesis of a small library using twelve halides and eight aldehydes resulted in the production of 74 desired compounds from 98 candidates, and were detected by their mass spectra. Antiproliferative effects of the crude compounds against HeLaS3 cells showed that eleven samples showed strong antitumor activity (IC50<0.05 microM). Further biological examination of four purified compounds by using five tumor cell lines (A549, HeLaS3, MCF7, TMF1, and P388) revealed strong cytotoxicity comparable to that of adriamycin.

New plastoquinones isolated from the brown alga, Sargassum micracanthum.

Four new plastoquinones were isolated from the methanolic extract of the brown alga, Sargassum micracanthum. Their structures were elucidated based on spectroscopic analysis and chemical conversions from 2-geranylgeranyl-6-methyl-1,4-benzohydroquinone. These plastoquinones exhibited significant antioxidant activities such as an inhibitory effect on lipid peroxidation and a radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH). Some of the new plastoquiones showed cytotoxic activity against cancer cell line.

Antioxidant and antiviral activities of plastoquinones from the brown alga Sargassum micracanthum, and a new chromene derivative converted from the plastoquinones.

Two plastoquinones were isolated from the methanolic extract of the brown alga Sargassum micracanthum, and these were identified as a known 2-geranylgeranyl-6-methylbenzoquinone and its hydroquinone, respectively, based on spectroscopic analysis. The absolute configuration of the secondary hydroxyl group was determined by the modified Mosher's method using the new chromene derivative converted from plastoquinones. One of the plastoquinones and the chromene exhibited significant antioxidant activities, such as an inhibitory effect on lipid peroxidation and a radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH). The benzoquinone-type compound and the chromene derivative were found to have potent antiviral activity against human cytomegalovirus (HCMV).

Baicalein 5,6,7-trimethyl ether, a flavonoid derivative, stimulates fatty acid beta-oxidation in skin fibroblasts of X-linked adrenoleukodystrophy.

The purpose of the present study is to identify bioactive compounds with potential for X-linked adrenoleukodystrophy (X-ALD) pharmacological therapy. Various plant natural products including flavonoids were tested for their ability to ameliorate the abnormality of very long chain fatty acid (VLCFA) metabolism in cultured skin-fibroblasts from X-ALD patients. Of the compounds tested, baicalein 5,6,7-trimethyl ether (baicalein-tri-Me) was found to significantly stimulate the VLCFA beta-oxidation activity. Furthermore, the incorporation of [1-(14)C]lignoceric acid into cholesteryl esters was markedly reduced towards the normal level and the VLCFA (C24:0 and C26:0) content was decreased. These results make baicalein-tri-Me a candidate for the therapeutic compound for X-ALD.

Effective irreversible alkylating reagents based on the structure of clavulones.

We describe the design and synthesis of alkylating reagents based on the structure of clavulones. They are composed of cross-conjugated dienone system and irreversibly reacted with two nucleophiles under mildly basic conditions via beta-elimination. Hydroxyl derivative 7b showed the highest reactivity toward thiols and showed the strongest cytotoxicity in Hela S3 cells among the three derivatives having a different protecting group at the tert-hydroxyl group.

Efficient solid-phase synthesis of clavulones via sequential coupling of alpha- and omega-chains.

We describe an efficient solid-phase synthesis of clavulones via the sequential coupling of the alpha- and omega-chains, involving two separate carbon-carbon bond-forming steps. The tetrahydropyranyl linker survived these reaction conditions and was cleaved without decomposing the unstable cross-conjugated dienones. Our methodology has allowed us to prepare six clavulone derivatives that are varied within the alpha-chain.

New marine diterpenoids from the Okinawan soft coral Clavularia koellikeri.

Chemical investigation of the Okinawan soft coral Clavularia koellikeri resulted in the isolation of two new cembrane diterpenoids (1 and 2) and one new dollabelane diterpenoid, 3. Their structures were determined on the basis of the results of spectroscopic analysis. Compounds 1 and 3 were examined for in vitro growth-inhibition effects toward tumor cells.

New biologically active marine sesquiterpenoid and steroid from the okinawan sponge of the genus Axinyssa.

A new bisabolane-type sesquiterpenoid, (E)-3-isocyanobisabolane-7,10-diene (1), and a new epidioxyergostane-type steroid, 9(11)-dehydroaxinysterol (2), were isolated from the Okinawan sponge of the genus Axinyssa. Their structures were elucidated based on the results of spectroscopic analysis and chemical conversion. Epidioxysterol 2 was found to show significant growth inhibitory effects against human cancer cell lines.

Tricycloclavulone and clavubicyclone, novel prostanoid-related marine oxylipins, isolated from the Okinawan soft coral Clavularia viridis.

Two novel prostanoid-related marine oxylipins, tricycloclavulone (1) and clavubicyclone (2), were isolated from the Okinawan soft coral Clavularia viridis. The structures of 1, having a tricyclo[5.3.0.0(1,4)]decane ring system, and 2, having a bicyclo[3.2.1]octane ring system, were elucidated on the basis of spectroscopic analysis. Clavubicyclone showed a moderate growth inhibition activity against tumor cells in vitro.