New antibacterial and 5-lipoxygenase activities of synthetic benzyl phenyl ketones: Biological and docking studies

We investigated twelve benzyl phenyl ketone derivatives which are synthetic precursors of isoflavonoids that are shown be good 5-hLOX inhibitors, especially those that have the catechol group, but these precursors never have been assayed as 5-hLOX inhibitors being a novelty as inhibitors of the enzyme, due to sharing important structural characteristics. Screening assays, half maximal inhibitory concentration (IC₅₀) and kinetic assays of all the studied molecules (5 µg/ml in media assay) showed that 1-(2,4-dihydroxy-3-methylphenyl)-2-(3-chlorophenyl)-ethanone (K205; IC₅₀ = 3.5 µM; K_i = 4.8 µM) and 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-nitrophenyl)-ethanone (K206; IC₅₀ = 2.3 µM; K_i = 0.7 µM) were potent, selective, competitive and nonredox inhibitors of 5-hLOX. Antioxidant behavior was also assayed by DPPH, FRAP, and assessing ROS production, and those with antibacterial and antiproliferative properties relating to 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-chlorophenyl)-ethanone (K208) established it as the most interesting and relevant compound studied, as it showed nearly 100% inhibition of bacterial growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Finally, docking studies were done that helped to characterize how the inhibitor structures correlated to decreased 5-hLOX activity.     

1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a K_m of 20 μm with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μm or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.     

A study by means of lactone inhibition of the role of a “half-chair” glycosyl conformation at the active centre of amylolytic enzymes

The kinetics of inhibition of porcine-pancreatic alpha amylase, sweet-potato beta amylase, and Aspergillus niger glucamylase enzymes have been studied by use of d-glucono-l,5-lactone and maltobiono-1,5-lactone as transition-state analogs. With d-glucono-1,5-lactone, alpha amylase can be inhibited, to a degree, non-competitively (K_i0.81mM,β≈e0.2), whereas with maltobionolactone, the inhibition is competitive (K_i10.31mM). The effect of beta amylase can be inhibited with maltobionolactone in a completely competitive way (K_iO.11mM), whereas with d-gluconolactone the inhibition is very poor (K_i21mM). Glucoamylase cannot be inhibited with maltobionolactone, whereas with d-gluconic acid, a completely mixed inhibition way be observed (K_i1.3mM). The ratio of the binding affinity of the lactones, products, and substrates, permits the conclusion that ring distortion takes place in the transition state with all three enzymes.     

Subcellular Localization and Properties of L-Galactono-γ-lactone Dehydrogenase in Spinach Leaves

L-Galactono-γ-lactone dehydrogenase, which catalyzes the final step of the biosynthesis of L-ascorbate, is bound to spinach mitochondrial membrane, as confirmed by linear sucrose density gradient centrifugation. The solubilized enzyme was very labile, but stabilized in the presence of L-galactono-γ-lactone under anaerobic conditions. The enzyme reduced cytochrome c and phenazine methosulfate in the presence of L-galactono-γ-lactone, but not when L-gulono-γ-lactone was used as an electron donor. The K_ms of the enzyme for L-galactono-γ-lactone and cytochrome c were 192 μM and 180 μM, respectively.     

Catalytic inhibition of gamma-aminobutyric acid - alpha-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog.

γ-Aminobutyric acid-α-ketoglutarate transaminase from $\text{Pseudomonas fluorescens}$ is irreversibly inhibited by 4-aminohex-5-yhoic acid, a new structural analog of GABA. The fact that this inhibition requires the pyridoxal form of the holoenzyme, and the formation of a Michaelis complex is in support of a catalytic mechanism. The compound is also active $\text{in vitro}$ and $\text{in vivo}$ on the same enzyme from mammalian brain.     

Degradation of L-arabonate by extracts of different filamentous fungi

Ten Aspergilli and Penicillia were tested for the capabilities of their extracts in degrading L-arabinose or L-arabonate nonphosphorolytically. L-arabonate dehydratase was nearly absent, while the reverse reaction of 2-keto-3-deoxy-L-arabonate (KDA) aldolase was operative in extracts of all the tested organisms grown on L-arabinose or L-arabonate as the sole carbon source. Degradation of different related substrates by cell-free extracts of Aspergillus ustus showed that L-arabonate, D-gluconate, D-galactonate and D-galactonic acid-γ-lactone were degraded under these conditions. Chromatographic studies identified the L-arabonate degrading products of such degradation in A. ustus as KDA (traces), pyruvic acid and α-ketoglutaric acid.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

Microbial transformation of ursolic acid by Syncephalastrum racemosum (Cohn) Schroter AS 3.264

Biotransformation of ursolic acid by the filamentous fungus Syncephalastrum racemosum (Cohn) Schroter AS 3.264 yielded five metabolites. Their structures were identified as 3β,21β-dihydroxy-urs-11-en-28-oic acid-13-lactone, 3β,7β,21β-trihydroxy-urs-11-en-28-oic acid-13-lactone, 1β,3β-dihydroxy-urs-12-en-21-one-28-oic acid, 1β,3β,21β-trihydroxy-urs-12-en-28-oic acid and 11,26-epoxy-3β,21β-dihydroxy-urs-12-en-28-oic acid based on NMR and MS spectroscopic analyses. The condensation reactions to form 28-oic acid-13-lactone ring and 11,26-epoxy ring are not frequently seen for the biotransformation of triterpenoids. One compound showed moderate inhibitory activity against protein tyrosine phosphatase 1B (PTP1B).     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Microbial Hydroxylation of (−)-α-Santonin by Aspergillus niger

The microbiological transformation of a sesquiterpene lactone, (−)-α-santonin was carried out by using Aspergillus niger MIL 5024 and MIL 5025. Strain MIL 5024 brings about the transformation of (−)-α-santonin (400 mg) to 11-hydroxy-(−)-α-santonin (P1) (50.4 mg), 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (P2) (22.4 mg), and 3,6-dihydroxy-9,10-seco-selina-1,3,5(10)-trien-9,12-dioic acid-12,6-lactone (P3) (27.1 mg), which were isolated and characterized by UV, IR, mass and ¹H-NMR spectroanalyses. All of these products are described for the first time. Results similar to those with MIL 5024 were also obtained from the transformation of (−)-α-santonin by the other isolate, strain MIL 5025.     

Reversible modification of amino groups in aspartate aminotransferase.

Amino groups in the pyridoxal phosphate, pyridoxamine phosphate, and apo forms of pig heart cytoplasmic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC .2.6.1.1) have been reversibly modified with 2,4-pentanedione. The rate of modification has been measured spectrophotometrically by observing the formation of the enamine produced and this rate has been compared with the rate of loss of catalytic activity for all three forms of the enzyme. Of the 21 amino groups per 46 500 molecular weight, approx. 16 can be modified in the pyridoxal phosphate form with less than a 50% change in the catalytic activity of the enzyme. A slow inactivation occurs which is probably due to reaction of 2,4-pentanedione with the enzyme-bound pyridoxal phosphate. The pyridoxamine phosphate enzyme is completely inactivated by reaction with 2,4-pentanedione. The inactivation of the pyridoxamine phosphate enzyme is not inhibited by substrate analogs. A single lysine residue in the apoenzyme reacts approx. 100 times faster with 2,4-pentanedione than do other amino groups. This lysine is believed to be lysine-258, which forms a Schiff base with pyridoxal phosphate in the holoenzyme.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Isolation and identification of novel sulfur-containing metabolites of spironolactone (Aldactone®)

In the urine of subjects given an oral dose of spironolactone [3-(3-oxo-7α-acetylthio-17β-hydroxy-4-androsten-17α-y1)propionic acid γ-lactone], six metabolites have been detected. One of the major metabolites was found to be the previously characterized de-thioacetylated compound, 3-(3-oxo-17β-hydroxy-4,6-androstadien-17α-y1)propionic acid γ-lactone (canrenone). Besides this a new major sulfur-containing metabolite has been isolated and identified as 3-(3-oxo-7α-methylsulfinyl-6β,17β-dihydroxy-4-androsten-17α-y1)propionic acid γ-lactone. This structural assignment was based on detailed analysis of its IR, NMR and UV spectra as well as comparison of its physical constants and chromatographic (TLC and GLC) characteristics with a synthetic sample. The three minor metabolites were found to be very labile and were readily converted to canrenone.     

Fungal hydroxylation of (−)-santonin and its analogues

Santonin (1) was incubated with separate growing cultures of Aspergillus niger ATCC 9142, Mucor plumbeus ATCC 4740, Whetzelinia sclerotiorum ATCC 18687, Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. Three novel metabolites were isolated: 11β,13-dihydroxysantonin (3), 6,7-dehydosantonin (5) and 3,6-dihydroxy-9-keto-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (7). 11β-Hydroxysantonin (2), 14-hydroxysantonin (4) and 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (6) were also isolated. Hydroxylation at C-9 followed by a retro-aldol reaction was postulated to have produced 6 and 7. Through the synthesis and fermentation of the santonin analogues: tetrahydrosantonin (8) and α-desmotroposantonin (12), several new compounds were obtained; the most significant being 9-keto-desmotroposantonin (14), which was indicative of C-9 monohydroxylation.     

Inhibition of glucuronokinase by substrate analogs

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a K_i value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl α-glucuronoside, methyl β-glucuronoside, β-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The β-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.     

Synthesis of a biologically active isomer of kotalanol,a naturally occurring glucosidase inhibitor

The syntheses of an isomer of kotalanol, a naturally occurring glucosidase inhibitor, and of kotalanol itself are described. The target compounds were synthesized by nucleophilic attack of PMB-protected 1,4-anhydro-4-thio-d-arabinitol at the least hindered carbon atom of two 1,3-cyclic sulfates, which were synthesized from d-mannose. Methoxymethyl ether and isopropylidene were chosen as protecting groups. The latter group was critical to ensure the facile deprotection of the coupled products in a one-step sequence to yield kotalanol and its isomer. The stereoisomer of kotalanol, with the opposite stereochemistry at the C-6′ stereogenic centre, inhibited the N-terminal catalytic domain of intestinal human maltase glucoamylase (ntMGAM) with a K_i value of 0.20 ± 0.02 μM; this compares to a K_i value for kotalanol of 0.19 ± 0.03 μM. The results indicate that the configuration at C-6′ is inconsequential for inhibitory activity against this enzyme.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.