The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Reductions of 2-enals, dehydrogenation of saturated aldehydes and their racemisation

Enoate reductase or clostridia containing this enzyme (Clostridium tyrobutyricum or C. kluyveri) catalyse the reduction of alpha,beta-unsaturated aldehydes (enals). The enantiomeric purity of the saturated aldehydes obtained from alpha-substituted enals is usually rather low and depends heavily on the reaction conditions. The reduction of the corresponding allyl alcohols to the saturated alcohols leads to much higher enantiomeric purities, though the reduction of the enal corresponding to the allyl alcohol to the saturated aldehyde is an intermediary step in the reaction sequence allyl alcohol----saturated alcohol. The explanation seems to be the racemisation of saturated aldehydes caused by enoate reductase. This is illustrated by the reduction of (E)-2-methylcinnamyl aldehyde to (R)-2-methyl-3-phenylpropanal or (R)-2-methyl-3-phenylpropanol under different conditions and measuring the racemisation of the aldehyde as well as the hydrogen-deuterium exchange of 3-phenylpropanal. In contrast to saturated carboxylates saturated aldehydes can be dehydrogenated to alpha,beta-unsaturated aldehydes (enals) by enoate reductase in the presence of electron acceptors such as oxygen or dichlorophenol indophenol. Under these conditions enoate reductase shows in the presence of oxygen a surprisingly high half life (greater than 20 h) as compared to that which is observed when the enzyme was used as a reductase with NADH in the presence of oxygen. In this case the enzyme is inactivated within a few minutes.     

A quinone-hydroquinone couple from the Brown alga Cystoseira stricta

2′E,6′E)-2-(10′,11′-Dihydroxygeranylgeranyl)-6-methylquinol and 2′E,6′E)-2(10′,11′-dihydroxyeranylgeranyl)-6-methyl-1,4-benzoquinone have been isolated from the brown alga Cystoseira stricta. The structures of the new algal metabolites have been elucidated by spectral analysis and chemical degradation.     

Electrical Properties of the Plasmalemma and Tonoplast in Valonia ventricosa

Studies were made on the electric potentials of the plasmalemma (E_co) and tonoplast (E_vc) in small cells (1-3 mm diameter) of Valonia ventricosa. To measure E_co, microelectrodes with long tapers were inserted into the vacuole with the path of electrode entry off-center. The microelectrode then was pushed across the vacuole and into the cytoplasm on the opposite side of the cell. A reference electrode was placed in the artificial seawater bathing the cell. A similar method was used to measure E_vc except that the reference electrode was placed in the vacuole.     

The correlation of cathodic peak potentials of vitamin K3 derivatives and their calculated electron affinities : The role of hydrogen bonding and conformational changes

2-methyl-1,4-naphtoquinone 1 (vitamin K₃, menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E_1/2) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E_A). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E_A) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.     

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.     

Stress-induced formation of echinatin and a metabolite, 5′-prenyl-licodione,in cultured Glycyrrhiza echinata cells

The production of a retrochalcone, echinatin, by isoflavone-rich Glycyrrhiza echinata (M-2) cultured cells was stimulated by the addition of yeast extract or calcium alginate beads to the culture medium. Combined addition of yeast extract and cycloheximide suppressed the formation of retrochalcone, suggesting de novo synthesis. A new metabolite was isolated from the induced cells and its structure was determined to be 1-[2,4-dihydroxy-5-(3-methyl-2-butenyl)phenyl]-3-(4-hydroxyphenyl)-1,3-propanedione (5′-prenyl-licodione).     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Effect of the half wave potential and change of some viologens on the rate of conversion of crotonic acid into butyric acid by enoate reductase

Summary The rate of the bio-electrochemical conversion of crotonic acid into butyric acid by enoate reductase is dependent on the type of viologen used. This illustrates that the reaction between enzyme and mediator, rather than the reaction between enzyme and crotonic acid, is rate limiting. Thus for bio-electrochemical conversion of enoates into saturated chiral acids immobilization of enoate reductase is beneficial from a kinetic point of view. The highest rate constant (k'=7.0×10⁶ M⁻¹.s⁻¹) was measured using mono-N-(aminopropyl) viologen.     

Synthesis and HMG-CoA reductase inhibition of 2-cyclopropyl-4-thiophenyl-quinoline mevalonolactones

A novel series of 2-cyclopropyl-4-thiophenyl quinoline-based mevalonolactones were synthesized from the substituted anilines by several reactions. Among them, (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(4-fluoro-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1d), (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1f) and (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4,7-di(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1q) showed potent HMG-CoA reductase inhibitory activity comparable with pitavastatin.     

Conversion of (2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid to xanthoxin acid by Cercospora cruenta, a fungus producing (+)-abscisic acid

(±)-(2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid was metabolized by Cercospora cruenta, which has the ability to produce (+)-abscisic acid (ABA), to give (±)-(2Z,4E)-xanthoxin acid, (±)-(2Z,4E)-5′-hydroxy-1′,2′-epoxy-1′,2′-dihydro-β-ionylideneacetic acid, (±)-1′,2′-epoxy-1′,2′-dihydro-β-ionone and trace amounts of ABA.     

Asymmetric reduction of activated alkenes using an enoate reductase from <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis>

A recombinant enoate reductase from Gluconobacter oxydans was heterologously expressed, purified, characterised and applied in the asymmetric reduction of activated alkenes. In addition to the determination of the kinetic properties, the major focus of this work was to utilise the enzyme in the biotransformation of different interesting compounds such as 3,5,5-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) and (E/Z)-3,7-dimethyl-2,6-octadienal (citral). The reaction proceeded with excellent stereoselectivities (>99% ee) as well as absolute chemo- and regioselectivity, only the activated C═C bond of citral was reduced by the enoate reductase, while non-activated C═C bond and carbonyl moiety remained untouched. The described strategy can be used for the production of enantiomerically pure building blocks, which are difficult to prepare by chemical means. In general, the results show that the investigated enoate reductase is a promising catalyst for the use in asymmetric C═C bond reductions.     

(E)-2,4-Bis(p-hydroxyphenyl)-2-butenal inhibits tumor growth via suppression of NF-κB and induction of death receptor 6

The Maillard reaction products are known to be effective in chemoprevention. Here, we focused on the anti-cancer effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal on in vitro and in vivo colon cancer. We analysed the anti-cancer activity of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal on colon cancer cells by using cell cycle and apoptosis analysis. To elucidate it’s mechanism, NF-κB DNA binding activity, docking model as well as pull-down assay. Further, a xenograft model of colon cancer was studied to test the in vivo effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal. (E)-2,4-Bis(p-hydroxyphenyl)-2-butenal inhibited colon cancer cells (SW620 and HCT116) growth followed by induction of apoptosis in a concentration-dependent manner via down-regulation of NF-κB activity. In docking model as well as pull-down assay, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal directly binds to three amino acid residues of IKKβ, thereby inhibited IKKβ activity in addition to induction of death receptor 6 (DR6) as well as their target apoptotic genes. Finally, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal suppressed anchorage-independent cancer cell growth, and tumor growth in xenograft model accompanied with apoptosis through inhibition of IKKβ/NF-κB activity, and overexpression of DR6. These results suggest that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal inhibits colon cancer cell growth through inhibition of IKKβ/NF-κB activity and induction of DR6 expression.     

Electroenzymatic synthesis of (S)-styrene oxide employing zinc oxide/carbon black composite electrode

(S)-styrene oxide, a useful chiral intermediate, has been synthesized using an electroenzymatic method with direct electrochemical FADH₂ regeneration. Low electroenzymatic efficiency arising from the fast FADH₂ reoxidation could be overcome by employing a zinc oxide/carbon black composite electrode. The attractive interaction between zinc oxide and styrene monooxygenase kept the local enzyme concentration high near the electrode surface, thereby increasing the accessibility of FADH₂ from the electrode surface to the enzyme. By adjusting the reaction conditions such as oxygen solubility, a high electroenzymatic efficiency of 65% was obtained. As a result, the reaction rate was increased while the amount of the side-products from the cofactor reoxidation process was decreased. The metal oxide/carbon black composite electrode can be efficiently used for electroenzymatic syntheses using diffusible-flavin dependent monooxygenases.     

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.     

Obtusastyrene and obtustyrene,cinnamylphenols from Dalbergia retusa

The heartwood of Dalbergia retusa contains, in addition to 8-O-methylretusin, (R)-4-methoxydalbergione, (R)-obtusaquinol and (+)-obtusafuran [(2R,3R)-2-phenyl-3-methyl-2,3-dihydro-5-hydroxy-6-methoxybenzofuran], the cinnamylphenols obtusastyrene [E-1-(4-hydroxybenzyl)-2-phenylethylenel, obtustyrene [E-1-(4-hydroxy-2-methoxybenzyl)-2-phenylethylene] and obtusaquinone [styryl-5-hydroxy-2-methoxy-4-quinonemethide]. The structural determination of compounds relied on spectra, degradations and syntheses.     

Purification and Characterization of Monovalent Cation-Activated Levodione Reductase from Corynebacterium aquaticum M-13

(6R)-2,2,6-Trimethyl-1,4-cyclohexanedione (levodione) reductase was isolated from a cell extract of the soil isolate Corynebacterium aquaticum M-13. This enzyme catalyzed regio- and stereoselective reduction of levodione to (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol). The relative molecular mass of the enzyme was estimated to be 142,000 Da by high-performance gel permeation chromatography and 36,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required NAD⁺ or NADH as a cofactor, and it catalyzed reversible oxidoreduction between actinol and levodione. The enzyme was highly activated by monovalent cations, such as K⁺, Na⁺, and NH₄⁺. The NH₂-terminal and partial amino acid sequences of the enzyme showed that it belongs to the short-chain alcohol dehydrogenase/reductase family. This is the first report of levodione reductase.     

Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

A monoterpene -lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.     

Antiparasitic activity of prenylated benzoic acid derivatives from Piper species

Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded three prenylated hydroxybenzoic acids, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid, 3-[(2E,6E,10E)-11-carboxy-13-hydroxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl]-4,5-dihydroxybenzoic acid and 3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-trimethyl-2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid, along with the known compounds, 4,5-dihydroxy-3-(E,E,E-11-formyl-3,7,15-trimethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (arieianal), 3,4-dihydroxy-5-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 4-hydroxy-3-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid, 4-hydroxy-3-(3,7-dimethyl-2,6-octadienyl)benzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid. Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. Riguera ester reactions and optical rotation measurements established the compounds as racemates. The antiparasitic activity of the compounds were tested against three strains of Leishmania spp., Trypanosoma cruzi and Plasmodium falciparum. The results showed that 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid exhibited potent and selective activity against L. braziliensis (IC₅₀ 6.5 μg/ml), higher that pentamidine used as control. Moreover, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl- 2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid showed moderate antiplasmodial (IC₅₀ 3.2 μg/ml) and trypanocidal (16.5 μg/ml) activities, respectively.