Enzymatic synthesis of stable, odorless, and powdered furanone glucosides by sucrose phosphorylase

Sucrose phosphorylase from Leuconostoc mesenteroides catalyzed transglucosylation from sucrose to 4-hydroxy-3(2H)-furanone derivatives. When 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone or 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (EHMF) were used as acceptors, their transfer ratios were more than 45%. In the case of glucosylation of HDMF, the major transfer product was identified as 2,5-dimethyl-3(2H)-furanone 4-O-alpha-D-glucopyranoside (DMF-G). In the case of glucosylation of EHMF, two major transfer products were obtained, and their structures were identified as 2-ethyl-5-methyl-3(2H)-furanone 4-O-alpha-D-glucopyranoside (2E5MF-G) and 5-ethyl-2-methyl-3(2H)-furanone 4-O-alpha-D-glucopyranoside (5E2MF-G) on the bases of spectrometric investigations. These glucosides were more stable than each aglycone. The glucosylated HDMF, DMF-G, was an odorless chemical, on the other hand, HDMF had a pineapple flavor. The glucosylated EHMF (EMF-G) were white odorless powders, though aglycone EHMF was a pale yellow syrup like a caramel with an intense sweet odor. Although DMF-G and EMF-G showed little radical-scavenging activity, hydrolyzates of these glucosides by an intestinal acetone powder from pigs had antioxidative activity as well as their aglycones. It was suggested that these glucosides improved some physical properties and may become prodrugs by glucosylation.     

Biosynthesis of flavour-active furanones by Saccharomyces cerevisiae during fermentation depends on the malt type used in medium preparation

Extracts of a coloured malt contained 4-hydroxy-5-monomethyl-3(2H)-furanone (HMMF), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) but not 4-hydroxy-5(or 2)-ethyl-2(or 5)-methyl-3(2H)-furanone (HEMF). Extracts of a pale malt did not contain any of the furanones. HMMF and HDMF were produced by Saccharomyces cerevisiae during fermen-tation of both types of malt extract. About 0.09 mg HEMF l ⁻¹ was synthesised during fermentation of the coloured malt extract but none was produced with the pale malt extract. Final concentrations of HDMF (2.0 mgl ⁻¹) and HEMF (0.09 mgl ⁻¹) were in excess of their aroma threshold values in water (0.16 and 0.02 mgl ⁻¹ respectively) after fermen-tation of the coloured malt extract. This revised version was published online in November 2006 with corrections to the Cover Date.     

FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase

The flavor of strawberry (Fragaria x ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of approximately 37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria x ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.     

New insights into the operative network of FaEO,an enone oxidoreductase from <Emphasis Type="Italic">Fragaria x ananassa</Emphasis> Duch.

The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.     

Chromones from the tubers of Eranthis cilicica and their antioxidant activity

Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of enzymatic hydrolysis allowed the chemical structures of the compounds to be assigned as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 5,7-dihydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 7-[(β-d-glucopyranosyl)oxy]-5-hydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 7-[(β-d-glucopyranosyl)oxy]-5-hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 9-[(O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, and 7-[(O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)oxy]methyl-4-hydroxy-5H-furo[3,2-g][1]benzopyran-5-one, respectively. The isolated compounds were evaluated for their antioxidant activity.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

Microbial transformation of 2-amino-4-methyl-3-nitropyridine

Biotransformation of the highly substituted pyridine derivative 2-amino-4-methyl-3-nitropyridine by Cunninghamella elegans ATCC 26269 yielded three products each with a molecular weight of 169?Da which were identified as 2-amino-5-hydroxy-4-methyl-3-nitropyridine, 2-amino-4-hydroxymethyl-3-nitropyridine, and 2-amino-4-methyl-3-nitropyridine-1-oxide. Biotransformation by Streptomyces antibioticus ATCC 14890 gave two different products each with a molecular weight of 169?Da; one was acid labile and converted to the other stable product under acidic conditions. The structure of the stable product was established as 2-amino-4-methyl-3-nitro-6(1H)-pyridinone, and that of the less stable product was assigned as its tautomer 2-amino-6-hydroxy-4-methyl-3-nitropyridine. Four of the five biotransformation products are new compounds. Several strains of Aspergillus also converted the same substrate to the lactam 2-amino-4-methyl-3-nitro-6(1H)-pyridinone. Microbial hydroxylation by C. elegans was found to be inhibited by sulfate ion. In order to improve the yield and productivity of the 5-hydroxylation reaction by C. elegans, critical process parameters were determined and Design of Experiments (DOE) analyses were performed. Biotransformation by C. elegans was scaled up to 15-l fermentors providing 2-amino-5-hydroxy-4-methyl-3-nitropyridine at ca. 13?% yield in multi-gram levels. A simple isolation process not requiring chromatography was developed to provide purified 2-amino-5-hydroxy-4-methyl-3-nitropyridine of excellent quality.     

Two new coumarins in Boenninghausenia albiflora

Two new isomeric coumarins were isolated from leaves of Boenninghausenia albiflora Reichb. Their structures were elucidated as (E)-7-hydroxy-6-(3-hydroxy-3-methyl-1-butenyl)-2(H)-1-benzpyran-2-one and (Z)-7-hydorxy-6-(3-hydroxy-3-methyl-1-butenyl)-2(H)-1-benzopyran-2-one.     

Insights into electron flux through manipulation of fermentation conditions and assessment of protein expression profiles in Clostridium thermocellum

While annotation of the genome sequence of Clostridium thermocellum has allowed predictions of pathways catabolizing cellobiose to end products, ambiguities have persisted with respect to the role of various proteins involved in electron transfer reactions. A combination of growth studies modulating carbon and electron flow and multiple reaction monitoring (MRM) mass spectrometry measurements of proteins involved in central metabolism and electron transfer was used to determine the key enzymes involved in channeling electrons toward fermentation end products. Specifically, peptides belonging to subunits of ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase (NFOR) were low or below MRM detection limits when compared to most central metabolic proteins measured. The significant increase in H₂ versus ethanol synthesis in response to either co-metabolism of pyruvate and cellobiose or hypophosphite mediated pyruvate:formate lyase inhibition, in conjunction with low levels of ferredoxin-dependent hydrogenase and NFOR, suggest that highly expressed putative bifurcating hydrogenases play a substantial role in reoxidizing both reduced ferredoxin and NADH simultaneously. However, product balances also suggest that some of the additional reduced ferredoxin generated through increased flux through pyruvate:ferredoxin oxidoreductase must be ultimately converted into NAD(P)H either directly via NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase (NfnAB) or indirectly via NADPH-dependent hydrogenase. While inhibition of hydrogenases with carbon monoxide decreased H₂ production 6-fold and redirected flux from pyruvate:ferredoxin oxidoreductase to pyruvate:formate lyase, the decrease in CO₂ was only 20 % of that of the decrease in H₂, further suggesting that an alternative redox system coupling ferredoxin and NAD(P)H is active in C. thermocellum in lieu of poorly expressed ferredoxin-dependent hydrogenase and NFOR.     

Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.     

Genotoxic effects of the chlorinated hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-dichloro-5-hydroxy-2[5H]-furanone in Tradescantia micronucleus assays

This is the first report of clastogenic effects of chlorinated hydroxyfuranones (CHFs) in plants. Two byproducts of water chlorination, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-dichloro 5-hydroxy-2[5H]-furanone (MA) induced a dose dependent increase of micronuclei (MN) in pollen mother cells of Tradescantia when doses up to 100 μg MX and 500 μg MA were applied directly to the inflorescences. In contrast, exposure of the stems in aqueous solutions containing up to 1 mg/I MX and 10 mg/I MA did not cause a positive response.     

ZnCl2 catalyzed new coumarinyl-chalcones as cytotoxic agents

A new series of coumarin-yl-chalcone derivatives (3a-m) had been designed and synthesized through different reactions such as aromatic addition, cyclization and Claisen-Schmidt reactions in good yields (54–78%). 5-acetyl-4-(2-hydroxyphenyl) -6-methyl-3, 4-dihydropyrimidin-2(1H) -one (1) has been synthesized by multi-component one pot reaction of salicylaldehyde, methyl acetoacetate and urea, which was further reacted with malonic acid employing ZnCl₂ catalyst to yield 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3, 4-dihydropyrimidin-2(1H) -one (2). The title compounds (3a-m) were synthesised by reacting 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3, 4-dihydropyrimidin-2(1H)-one (2) with different aromatic aldehydes in the presence of potassium hydroxide. In silico studies, a preliminary screening method for predicting the anti-cancer activity was performed for the synthesized compounds (3a-m) against Src, Alb tyrosine kinase and homology model protein (PDB ID: 4csv). The derivatives 3h and 3m showed moderate binding energies. The in vitro cytotoxic activity was evaluated for the compounds 3h and 3m by using human cancer cell-line morphology and MTT assay against three human cell-lines A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast). The results indicate that the derivatives 3h and 3m display significant anti-cancer activity, however it was found to be less cytotoxic when compared to the standard used i.e. Imatinib.     

Effect of Some Xenobiotics on the Activities of Enzymes Relating to the Glucuronic Acid Pathway and on the Ascorbic Acid Metabolism in Guinea Pigs

An improvement to the aroma and taste of Burley cigarettes was achieved by adding volatiles from roasted, Japanese flue-cured tobacco. The addition of roasted tobacco volatiles was also found to decrease the offensive odor and taste. Among the fractions obtained from silica gel column chromatography of roasted tobacco volatiles, Fr. 8 (diethyl ether elution) exhibited the best improvement to the organoleptic properties of cigarette smoke. A great part of the improvement was attributable to 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 3-hydroxy-4,5-dimethyl-2(5H)-furanone, both found in small amounts in Fr. 8 but possessing a strong impact on the olfactory properties.     

Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development.     

Evaluation of insulin-mimetic activities of vanadyl and zinc(II) complexes from the viewpoint of heterocyclic bidentate ligands

Vanadyl sulfate (VOSO₄) has been clinically tested in diabetic patients since 1995. Oral administrations of VOSO₄ improved the type 2 diabetic state with respect to plasma glucose, HbA_1c, and fructosamine levels. The development of toxicity by increasing the administration of VOSO₄ should be avoided. One method was the utilization of vanadyl complexes with coordination compounds that are low-toxic and low-molecular-weight ligands to enhance the permeation of the metal ion to lipid bilayer membrane. Over a decade we have focused on a variety of heterocyclic compounds as bidentate ligands for metal ions. Vanadyl and zinc(II) complexes of 1-substituted 3-hydroxy-2-methyl-4(1H)-pyridinethiones, 4,5,6-substituted 1-hydroxy-2(1H)-pyrimidinones, 4-(p-substituted)phenyl-3-hydroxythiazole-2(3H)-thiones, 3-hydroxypyrone, 1-alkyl- or 1-phenylalkyl-3-hydroxy-2(1H)-pyridinethiones, optically active 1-substituted 3-hydroxy-4(1H)-pyridinethiones, and 5-dialkylsulfonamido- or 5,7-bis(dialkylsulfonamido)-8-hydroxyquinolines were prepared, and their insulin-mimetic activities were evaluated in terms of IC₅₀ values which stand for a 50% inhibitory concentration of the free fatty acid release from isolated rat adipocytes. In this article, the relationship between the insulin-mimetic activity and the partition coefficient, the chirality, the substituent effect, molecular weight, the pK_a value, and the coordination mode was discussed. In vivo blood glucose-lowering effects of the vanadyl complex with 1-hydroxy-4,6-dimethyl-2(1H)-pyrimidinone in streptozotocin (STZ)-induced diabetic rats and the zinc(II) complexes with 4-(p-chlorophenyl)thiazole- and 4-methylthiazole-2(3H)-thione in KK-A^y mice were also discussed.     

Clostridium scindens baiCD and baiH genes encode stereo-specific 7α/7β-hydroxy-3-oxo-Δ4-cholenoic acid oxidoreductases

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7α/β-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Δ⁴-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce α/β-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7β-hydroxy-3-oxo-Δ⁴-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Δ⁴-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7α-hydroxy-3-oxo-Δ⁴-cholenoic acid oxidoreductase.     

Chemical constituents from the fresh flower buds of Musa nana and their chemotaxonomic significance

Phytochemical study on the fresh flower of Musa nana Lour. provided seventeen known compounds including two alkaloids, 3-(hydroxyacetyl)-indole (1), bi-indol-3-yl (2), two terpenoids, 5-[(1R)-1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl]-3-methyl-, (2Z, 4E) −2, 4-pentadienoic acid (Valdes), 5, 6(S), 7, 7a(R)-tetrahydro-6-hydroxy-4,4-dimethyl-2(4H)-benzofuranone (4), seven phenols (5–11), three phenylphenalenones, 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one (12), 2-methoxy-9-phenyl-1H-phenalen-1-one (13), 2-methoxy-9-(4-methoxyphenyl)-1H-phenalen-1-one (14), and three lipids (15–17). In the present study, all the compounds were isolated for the first time from the species M. nana. Ten compounds including 1-8 and 15-16 have never been previously encountered in the Musaceae family. Furthermore, the chemotaxonomic significance of these isolates was also discussed.     

Structural and Kinetic Characterization of 4-Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolase,a Protocatechuate Degradation Enzyme Evolutionarily Convergent with the HpaI and DmpG Pyruvate Aldolases

4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate α-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k_cat/K_m and k_cat for the enzyme is consistent with a single deprotonation with pK_a values of 8.0 ± 0.1 and 7.0 ± 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered α-β-β-α sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199′. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.     

Chromone derivatives from Halenia elliptica and their anti-HBV activities

Nine water-soluble chromone derivatives, including chromone-2-carboxylic acids, 2-methylchromones and their structural hybrids, were isolated from aerial tissues of Halenia elliptica (Gentianaceae), six of which were previously unknown. Their structures were elucidated by comprehensive mass, 1D and 2D NMR spectroscopic analyses and chemical derivatization. Two unstable structural hybrids of chromone-2-carboxylic acids and 2-methylchromones, viz. 3-acetyl-8-hydroxy-4H-1-benzopyran-4-one-2-carboxylic acid (halenic acid C) and 2-(8-hydroxy-2-methyl-4H-1-benzopyran-4-one-3-yl)-2-oxoacetic acid (halenichromone A), were isomers and were interconvertible. The proposed mechanism of their acid-catalyzed isomerization in aqueous solvent is described. In addition, 2-methylchromones, 8-hydroxy-2-methyl-4H-1-benzopyran-4-one, and 8-methoxy-2-methyl-4H-1-benzopyran-4-one, were found to exhibit a strong inhibitory effect towards hepatitis B virus (HBV) in vitro without showing significant cytotoxicity.