Purification and cDNA Cloning of NADPH-Dependent Aldoketoreductase, Involved in Asymmetric Reduction of Methyl 4-Bromo-3-Oxobutyrate, from Penicillium citrinum IFO4631

Penicillium citrinum was found to catalyze the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate [(S)-BHBM] with high optical purity. From the strain, a cDNA clone encoding a novel NADPH-dependent alkyl 4-halo-3-oxobutyrate reductase (KER) was isolated. Escherichia coli cells overexpressing KER produced (S)-BHBM in the presence of an NADPH-regeneration system.     

Engineering of NADPH-dependent aldo-keto reductase from <Emphasis Type="Italic">Penicillium citrinum</Emphasis> by directed evolution to improve thermostability and enantioselectivity

Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system.     

Biocatalytic production of (S)-4-bromo-3-hydroxybutyrate and structurally related chemicals and their applications

The enzymatic production of (S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with (S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum β-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of (S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of (S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds.     

Purification and characterization of NADPH-dependent aldo–keto reductase specific for β-keto esters from Penicillium citrinum,and production of methyl (S)-4-bromo-3-hydroxybutyrate

A novel -keto ester reductase (KER) was purified to homogeneity from recombinant Escherichia coli (pTrcKER) cells, which efficiently expressed the ker gene cloned from Penicillium citrinum IFO4631. The enzyme was monomeric and had a molecular mass of 37 kDa. It catalyzed the reduction of some -keto esters, especially alkyl 4-halo-3-oxobutyrates. However, it did not catalyze the reverse reaction, the dehydrogenation of alkyl 4-halo-3-hydroxybutyrates and other alcohols. The enzyme required NADPH as a cofactor and showed no activity with NADH. Therefore, it was defined as a NADPH-dependent aldo–keto reductase (AKR3E1), belonging to the AKR superfamily. The enzyme stereospecifically produced methyl (S)-4-bromo-3-hydroxybutyrate from its keto derivative with high stereospecificity (97.9% enantiomer excess). E. coli cells expressing KER and glucose dehydrogenase in the water/butyl acetate two-phase system achieved a high productivity of (S)-4-bromo-3-hydroxybutyrate (277 mM, 54 mg/ml) in the organic solvent layer.     

Norepinephrine release in the heart atria of diabetic rats

The content, release and uptake of norepinephrine (NE) in the sympathetic nerves of the rat heart atria were studied in the course of diabetes and in age-matched controls. Diabetes was induced by streptozotocin (STZ) and rats were subjected to further experiments 1, 4 or 7 months later (STZ1, STZ4, STZ7). Isolated atria were superfused with oxygenated Krebs-Henseleit (KH) solution. After equilibration, four 10-min fractions were collected: B1, basal release of NE; S1, potassium-evoked release (KER), where NE outflow was stimulated by depolarisation with 50 mmol/l KCl; B2, basal release of NE under the influence of the neuronal uptake blocker desipramine (DES); S2, KER under the influence of DES. The content of NE was measured by radioimmunoassay. In STZ4 and STZ7 rats, NE concentrations were significantly lower in both atria compared to controls. B1 and S1 were significantly higher in STZ4 than in control atria. DES increased KER of NE in controls only. In contrast, DES caused a significant decrease in B2 and S2 in STZ4 atria, suggesting that a substantial portion of NE release was due to a calcium-independent carrier-mediated process. In experiments with calcium-free KH solution in fractions B2 and S2, KER ill controls was nearly abolished. However, in STZ4 and STZ7 atria, S2 was still significantly higher than B2. In conclusion, the NE-releasing mechanism may be different in the chronically diabetic animals than in healthy subjects and may contribute to the decreased NE concentration in the STZ atria.     

On the regioselectivity of the Hanessian-Hullar reaction in 4,6-O-benzylidene protected galactopyranosides

The N-bromosuccinimide mediated fragmentation of methyl 4,6-O-benzylidene-beta-D-galactopyranoside results in the formation of methyl 4-O-benzoyl-6-bromo-6-deoxy-beta-D-galactopyranoside and methyl 4-O-benzoyl-3-bromo-3-deoxy-beta-D-gulopyranoside, as opposed to the methyl 6-O-benzoyl-3-bromo-3-deoxy-beta-D-gulopyranoside originally reported. The kinetic methyl 4-O-benzoyl-6-bromo-6-deoxy-beta-D-galactopyranoside rearranges to the thermodynamic methyl 4-O-benzoyl-3-bromo-3-deoxy-beta-D-gulopyranoside under the reaction conditions, likely via a 3,6-anhydro galactopyranoside. The NBS-mediated cleavage of 4,6-O-benzylidene acetals in the galactopyranoside series is therefore shown to conform to the regiochemistry observed in the corresponding gluco- and mannopyranoside series with preferential cleavage of the C6-O6 bond by an ionic mechanism.     

Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex.

Highly purified branched-chain 2-oxo acid dehydrogenase complex (BCOADC) oxidizes 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with Km values of 67 microM and 18 microM respectively. The Vmax. for oxidation of these substrates is 27% and 53% respectively of that for 3-methyl-2-oxobutyrate. Highly purified pyruvate dehydrogenase complex (PDC) oxidizes 2-oxobutyrate (Km 100 microM; Vmax. 49% of that for pyruvate) but not 4-methylthio-2-oxobutyrate, whereas 2-oxoglutarate dehydrogenase complex will not utilize either 2-oxo acid as substrate. BCOADC kinase is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with half-maximal inhibition by 45 microM and 50 microM respectively. Phosphorylation of BCOADC in isolated adipocytes is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, consistent with their inhibitory action of BCOADC kinase. Phosphorylation of PDC is decreased by 2-oxobutyrate, but not by 4-methylthio-2-oxobutyrate.     

Synthesis of (S)-2-fluoro-L-daunosamine and (S)-2-fluoro-D-ristosamine

Derivatives of (S)-2-fluoro-L-daunosamine and (S)-2-fluoro-D-ristosamine were synthesized, starting ultimately from 2-amino-2-deoxy-D-glucose which was converted, according to the literature, into methyl 2-benzamido-4, 6-O-benzylidene-2-deoxy-3-O-(methylsulfonyl)-alpha-D-glucopyranoside (2). Treatment of 2 with tetrabutylammonium fluoride gave a 63% yield of (known) methyl 3-benzamido-4,6-O-benzylidene-2,3-dideoxy-2-fluoro-alpha-D-altropyran oside (4), together with a 6% yield of its 2-benzamido-2,3-dideoxy-3-fluoro-alpha-D-gluco isomer. From 4, the corresponding 6-bromo-2,3,6-trideoxyglycoside 4-benzoate (6) was obtained by Hanessian-Hullar reaction. Dehydrobromination of 6, followed by catalytic hydrogenation of the resulting 5-enoside, and subsequent debenzoylation and N-trifluoroacetylation, afforded the fluorodaunosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-beta-L-galactopyranos ide. Reductive debromination of 6, followed by debenzoylation and N-trifluoroacetylation, gave the fluororistosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-alpha-D-altropyran oside. The 1H-n.m.r. spectra of the new aminofluoro sugars are discussed with respect to the effects of neighboring amino and acylamido substituents on geminal and vicinal 1H-19F coupling constants, in comparison with the reported effects of oxygen substituents.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Synthesis of 5-hydroxy-2-(beta-D-ribofuranosyl)pyran-4-one from a pyranulose glycoside.

The synthesis of 5-hydroxy-2-(beta-D-ribofuranosyl)pyran-4-one (9) is described. Treatment of pyranulose glycoside with bromine in carbon tetrachloride afforded brompyranulose glycoside in 90% yield. The reaction of (6S)- and (6R)-4-bromo-6-hydroxy-6-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-6H- pyran-3-one (2) in acidic media was examined with the following results: the reaction of 2 with trifluoroacetic acid (TFA) in dioxane afforded a mixture of 5-hydroxy-2-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyran-4-one (3) and its furan derivative 5-hydroxy-2-{5-(benzoyloxy)methyl]furan-2-yl}pyran-4-one (4), but the use of hydrochloric acid formed the bromofurfural, 3-bromo-5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-2-furancarboxyal dehyde only. Acetylation of a mixture (3 and 4) with acetic anhydride facilitated product separation to give the corresponding acetates 5-acetoxy-2-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyran-4-one (5) and 5-acetoxy-2-{5-[(benzoyloxy)methyl]furan-2-yl}pyran-4-one (6). Treatment of 5 with hydrazine afforded 3-hydroxymethyl-6-(beta-D-ribofuranosyl)-1H-pyridazin-4-one in 43% yield. Debenzoylation of 5 with aq ammonia gave 9 in 50% yield.     

Retinoic acid metabolism inhibition by 3-azolylmethyl-1H-indoles and 2, 3 or 5-(alpha-azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or alpha-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl) (1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 microM) ketoconazole exerted similar inhibitory effect (70% inhibition).     

A nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate, irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase at micromolar concentrations

We previously showed that 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate inactivates cAMP phosphodiesterase (PDE3A); however, millimolar concentrations were needed to inactivate PDE3A because of ongoing hydrolysis. We have now synthesized a nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate (S(p)-8-BDB-TcAMPSMe). S(p)-8-BDB-TcAMPSMe inactivates PDE3A in a time-dependent, irreversible manner, exhibiting saturation kinetics with a k(max) of (19.5 +/- 0.3) x 10(-3) min(-1) and a K(I) of 3.5 +/- 0.3 muM. To ascertain whether S(p)-8-BDB-TcAMPSMe reacts in the active site, nonhydrolyzable analogues of the substrate cAMP, or the competitive inhibitor cGMP, were included to protect against the inactivation of PDE3A. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) values in micromolar) is as follows: S(p)-cAMPS (18) > R(p)-cGMPS (560) and S(p)-cGMPS (1260) > 5'-AMP (17 660), R(p)-cAMPS (30 110), and 5'-GMP (42 170). We docked S(p)-8-BDB-TcAMPSMe into PDE3A, based on the structural model of PDE3A-cAMP and the kinetic data from site-directed mutants. The S(p)-8-BDB-TcAMPSMe fits into the active site in the model. These results suggest that inactivation of PDE3A by the affinity reagent is a consequence of reaction at the overlap between cAMP and cGMP binding regions in the active site. S(p)-8-BDB-TcAMPSMe has proven to be an effective active site-directed irreversible cAMP affinity label for platelet PDE3A and can be used to identify amino acids in the active site of PDE3A as well as in other cAMP phosphodiesterases.     

Synthesis of (2S,2'R,3S,4R)-2-(2'-hydroxy-21'-methyidocosanoylamino)-1,3,4-pentadecanetriol,the ceramide sex pheromone of the female hair crab,Erimacrus isenbeckii

The ceramide sex pheromone [(2S,2'R,3S,4R)-2-(2'-hydroxy-21-methyldocosanoylamino)-1,3,4-pentadecanetriol (1)] of the female hair crab (Erimacrus isenbeckii) was synthesized by starting from (S)-serine and 12-bromo-1-dodecanol.     

Synthesis of 3-amino-2,3,6-trideoxy-D-ribo-hexose hydrochloride.

The title sugar, the 5-epimer of daunosamine, was prepared in a sequence of high-yielding steps from methyl alpha-D-mannopyranoside (1). Conversion of 1 into methyl 3-acetamido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-alpha-D-ribo-hexopyranoside (2), followed by reduction with hydrogen and Raney nickel, gave the 4-benzoate (3) of methyl 3-acetamido-2,3,6-trideoxy-alpha-D-ribo-hexopyranoside (4). Saponification of 3 gave 4 as an oil that gave a crystalline 4-acetate (8). N-Deacetylation of 4 was effected with barium hydroxide, and the resultant glycoside was hydrolyzed to give 3-amino-2,3,6-trideoxy-D-ribo-hexose hydrochloride (7). The 3-benzamido analogue (5) of 4 was prepared from 4 by N-deacetylation and subsequent benzoylation, and hydrolysis of 5 gave crystalline 3-benzamido-2,3,6-trideoxy-D-ribo-hexose (6). The crystalline 3-acetamido analogue (9) of 6 was obtained by acid hydrolysis of the glycoside 4.     

Analogue-based design, synthesis and molecular docking analysis of 2,3-diaryl quinazolinones as non-ulcerogenic anti-inflammatory agents

In our effort to identify potent gastric sparing anti-inflammatory agents, a series of methyl sulfanyl/methyl sulfonyl substituted 2,3-diaryl quinazolinones were designed by analogue-based design strategy and synthesized for biological evaluation. Subsequently, the compounds were evaluated for both cyclooxygenase inhibitions by ovine COX assay and carrageenan-induced rat paw edema assay. All the methyl sulfonyl substituted quinazolinones were exhibited promising anti-inflammatory activity. In particular, 6-bromo-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-quinazolin-4-one, 7-chloro-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-quinazolin-4-one, 3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-3H-quinazolin-4-one and 6-bromo-3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-3H-quinazolin-4-one emerged as the most active compounds in the series. The results of ulcerogenic activity assay suggest that these compounds are gastric safe compared to indomethacin. The molecular docking analysis was performed to understand the binding interactions of these compounds to COX-2 enzyme. The results from the present investigation suggests that 2,3-diaryl quinazolinones as a promising template for the design of new gastric safe anti-inflammatory agents, which can be further explored for potential anti-inflammatory activity.     

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.     

An application of the palladium-catalyzed,allylic amination of unsaturated sugars: a new synthesis of d-forosamine

Methyl 4-O-benzoyl-6-bromo-6-deoxy-α-d-glucopyranoside, obtainable from methyl 4,6-O-benzylidene-α-d-glucopyranoside (1), was converted into the 2,3-unsaturated 4-benzoate (3) by application of the triiodoimidazole method. Debenzoylation of 3, followed by acetylation, afforded crystalline methyl 4-O-acetyl-6-bromo-2,3,6-trideoxy-α-d-erythro-hex-2-enopyranoside (5). Treatment of 5 with benzylmethylamine under conditions of palladium-catalyzed, allylic substitution gave a separable mixture of the corresponding 4-(N-benzyl)methylamino-6-bromo-2-enoside (37%) and the 4,6-di-[(N-benzyl)methylamino]-2-enoside (55%). Debromination of 5 with lithium triethylborohydride, proceeding with simultaneous deacetylation, readily yielded methyl 2,3,6-trideoxy-α-d-erythro-hex-2-enopyranoside (8). The 4-acetate of 8 (obtained by reacetylation), and also its 4-benzoate (prepared by a different synthetic route), furnished high yields (～80%) of methyl 4-[(N-benzyl)-methylamino]-2,3,4,6-tetradeoxy-α-d-erythro-hex-2-enopyranoside (13) upon palladium-catalyzed animation with benzylmethylamine. Catalytic hydrogenation of 13 effected saturation of the alkenic double bond and removal of the N-benzyl group, to afford methyl 2,3,4,6-tetradeoxy-4-methylamino-α-d-erythro-hexopyranoside, which was subsequently N-methylated with formaldehyde and sodium borohydride, to give its N,N-dimethyl analog, methyl α-d-forosaminide (15). The overall yield of 15 from 1 was 24%. Hydrolysis of 15 to the free sugar has been described previously.     

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.     

<Emphasis Type="Italic">In Vitro</Emphasis> antiviral activity of red alga, <Emphasis Type="Italic">Polysiphonia morrowii</Emphasis> extract and its bromophenols against fish pathogenic infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus

Our previous investigation revealed that 80% methanolic extract of the red alga Polysiphonia morrowii has significant antiviral activities against fish pathogenic viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). The present study was conducted to identify compounds attributed for its antiviral activities and investigate their antiviral activities against IHNV and IPNV. Activity-guided fractionation for 80% methanolic extract of Polysiphonia morrowii using a cell-based assay measuring virus-induced cytopathic effect (CPE) on cells yielded a 90% methanolic fraction, which showed the highest antiviral activity against both viruses among fractions yielded from the extract. From the fraction, two bromophenols were isolated and identified as 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) based on spectroscopic analyses. For both compounds, the concentrations to inhibit 50% of flounder spleen cell (FSP cell) proliferation (CC₅₀) and each viral replication (EC₅₀) were measured. In the pretreatment test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihy-droxybenzaldehyde (2) exhibited significant antiviral activities showing selective index values (SI = CC₅₀/EC₅₀) of 20 to 42 against both IHNV and IPNV. In direct virucidal test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) showed significant antiviral activités against both viruses while 3-bromo-4,5-dihydroxybenzaldehyde (2) was significantly effective against only IHNV. Although antiviral efficacies of both compounds against IHNV and IPNV were lower than those of ribavirin used as a positive control, our findings suggested that the red alga Polysiphonia morrowii and isolated two bromophenols may have potential as a therapeutic agent against fish viral diseases.     

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.