Enzymatic cyclization of all-trans pentaprenyl and hexaprenyl methyl ethers by a cell-free system from the protozoon Tetrahymena pyriformis. The biosynthesis of scalarane and polycyclohexaprenyl derivatives

A cell-free system from the protozoon Tetrahymena pyriformis capable of cyclizing squalene into tetrahymanol cyclizes all-trans pentaprenyl methyl ether to a scalarane-type sesterterpene and all-trans hexaprenyl methyl ether to bicyclo-, tricyclo-, tetracyclo- and pentacyclohexaprenyl methyl ethers, each corresponding to a possible cationic intermediate. The structures of the cyclization products have been determined by spectroscopic methods and are compatible with a biogenetic scheme involving polyprenyl ether cyclization. This is the first direct proof of an enzymatic cyclization of higher isoprenic alcohol derivatives, and we assume it was performed by the squalene-to-hopane cyclase of the protozoon. The formation of a scalarane-type sesterterpene from C25 polyprenyl methyl ether suggests that these terpenoids, whose presence is restricted to a few sponges, might be in fact microbial metabolites. Tricyclopolyprenyl derivatives have been identified in the organic matter from numerous sediments and they were interpreted as being chemical fossils of still unidentified microorganisms. The cyclization of hexaprenyl methyl ether is the first attempt of identification of these tricyclopolyprenol derivatives in living organisms.     

Non-specific activation of mouse peritoneal macrophages by a freshwater ciliate, Tetrahymena pyriformis

Toxoplasma-killing activities of mouse peritoneal macrophages activated by the extracts of Tetrahymena pyriformis (Korean and Chinese strains) were evaluated, and the active protein fractions from both strains were partially characterized by a method including chromatographies and SDS-PAGE. The first peak in Korean strain and the second peak in Chinese strain of T. pyriformis obtained by DEAE-Sephadex A-50 chromatography were most effective in the activation of macrophages to kill Toxoplasma gondii tachyzoites in vitro. Subsequent fractionations of obtained peak fractions were performed on a Sephadex G-200 gel. The first peaks fractionated from both strains of T. pyriformis had the highest toxoplasmacidal activities, and when subjected to the SDS-PAGE, one prominent band was visualized for each of the strains showing the same molecular weight of ca. 52.6 kDa. This active protein is suggested to be related to non-specific activation of mouse peritoneal macrophages.     

Studies on the biosynthesis of tetrahymanol in Tetrahymena pyriformis. The mechanism of inhibition by cholesterol

Tetrahymanol biosynthesis by the protozoan Tetrahymena pyriformis was progressively inhibited by the inclusion of cholesterol in the growth medium. Studies with labelled precursors of tetrahymanol have established that there are two major sites of inhibition in whole cells. The inhibition at the first site, between acetate and mevalonate, occurred rapidly after addition of cholesterol. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), a predominantly cytosolic enzyme in this organism, was not inhibited in cholesterol-grown cells nor by addition of cholesterol directly to the assay medium. The second major site of inhibition in whole cells is between mevalonate and squalene and this is accompanied by inhibition of the enzyme that converts farnesyl-pyrophosphate into squalene (squalene synthetase). Squalene cyclase is partially inhibited. The conversion of mevalonate into tetrahymanol in vitro was not inhibited by the addition of cholesterol to the assay medium. Tetrahymanol added to the culture medium is taken up by the cells but does not inhibit endogenous biosynthesis. It is suggested that cholesterol inhibits the later stages of tetrahymanol biosynthesis by causing a change in membrane structure and function which alters the activity of membrane-bound enzymes.     

Effect of hexamethylene bisacetamide (HMBA) on the microtubular system of Tetrahymena pyriformis

HMBA (10-[3-hydroxy-4-methoxy benzylidene)]-9(10H)-anthracenone) is an inhibitor of tubulin polymerization and a developmental inducer in mammalian cells. The effect of HMBA on the microtubular system of Tetrahymena was investigated. This is the first case when its effect was studied in an unicellular animal, by using immunocytochemistry, confocal microscopy, Flutax-1 staining and flow cytometry. In Tetrahymena, HMBA (20 nM.; 10 and 45 min) significantly decreased the label of transversal microtubules (without affecting longitudinal ones) and also decreased the diffuse cytoplasmic fluorescence (tubulin-dimer pool). However, it increased the gross amount of alpha-tubulin and acetylated tubulin. Cilia showed an extraordinary strong labeling. Longer treatments (45 min) were toxic. There is a possibility, that the extremely rich tubulin content of cilia was due to the inducer effect of HMBA or to the self-defense of the cell.     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.     

In vivo metabolism of platelet-activating-factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the protozoan Tetrahymena pyriformis

The metabolism of exogenous platelet-activating-factor was studied in the protozoan Tetrahymena pyriformis in vivo. When the cells are exposed to 1.10(-6) M PAF, the molecule is rapidly metabolized to 1-O-alkyl-2-acyl(long chain)-GPC, a major component of the protozoan membranes. The appearance of lyso-PAF from the first minutes even in low levels provides evidence that deacetylation is an intermediate step. After incubation for 30 min, transformation to aminoethyl phosphonolipids is also observed. The fate of PAF in concentrations 1.5.10(-11) M or 1.10(-8) M PAF, was the same. An amount of PAF depending on the external PAF concentration remained intact in the cell even after 1 h incubation. Our results suggest that the easily cultured protozoan can be a useful model for studying PAF's metabolism.     

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.     

Separation by high-performance liquid chromatography of (3R)- and (3S)-beta-leucine as diastereomeric derivatives

For studies on the coenzyme B12-dependent enzyme, leucine-2,3-aminomutase, (3R)- and (3S)-beta-leucines were synthesized. The 10-camphorsulfonamide p-nitrobenzyl esters could be resolved by normal-phase HPLC. A much better separation was obtained by reversed-phase HPLC of the diastereomeric derivatives obtained by treatment of -leucine with Marfey's reagent (N2-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide).     

Short and efficient synthesis of (2S,3R,4R,5R) and (2S,3R,4R,5S)-tetrahydroxyazepanes via the Henry reaction

The Henry reaction with the easily available alpha-d-xylo-pentodialdose afforded a diastereomeric mixture of nitroaldoses with the alpha-d-gluco- and beta-l-ido-configuration, respectively, in good yield. When n-BuLi was used as the base, the reaction afforded the alpha-d-gluco-nitroaldose as the only product. The reduction of the nitro group in the alpha-d-gluco- and beta-l-ido-nitroaldoses, removal of the protecting groups and intramolecular reductive cyclo-amination afforded the corresponding (2S,3R,4R,5R) and (2S,3R,4R,5S) tetrahydroxyazepanes.     

Synthesis of (3R,10R)- and (3S,10S)-Diastereomers of 3,10-Dimethylspermine

Russian Journal of Bioorganic Chemistry - A simple and practical 10-step synthesis is reported for previously unknown diastereomers of C?methylated spermine (Spm) analogue,...     

Synthesis of (2S,3R)-beta-methyltyrosine catalyzed by tyrosine phenol-lyase.

A one-step enzymatic synthesis of the conformationally restrained tyrosine analog (2S,3R)-beta-methyltyrosine is reported. This synthesis extends the preparative chemistry associated with tyrosine phenol-lyase. This beta-methyltyrosine derivative was shown to be an efficient protein tyrosine kinase substrate, suggesting that conformational restraint may ultimately be used to enhance tyrosine kinase recognition of substrates.     

Effect of alternating magnetic fields (60--100 gauss, 60 Hz) on Tetrahymena pyriformis.

Tetrahymena pyriformis and neuroblastoma cells were studied following exposure to low intensity low frequency alternating magnetic fields. Tetrahymena showed cytomorphologic changes, with delayed and reduced cell division concurrent with increased oxygen uptake. The resulting dead cells appeared intact, as compared with dissolution characteristic of the control group. In contrast, magnetically exposed actively growing neuroblastoma cells showed no growth alterations in vitro, but were affected when exposed in vivo.     

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.     

An efficient system for the asymmetric acylation of (R,S)-3-n-butylphthalide catalyzed by novozyme 435

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran-hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee(S)), enantiomeric excess of product (ee(P)), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30 °C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee(S) and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.     

Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli.

The phosphoenolpyruvate mutase gene from Tetrahymena pyriformis has been cloned and overexpressed in Escherichia coli. To our knowledge, this is the first Tetrahymena gene to be expressed in E. coli, a task made more complicated by the idiosyncratic codon usage by Tetrahymena. The N-terminal amino acid sequence of phosphoenolpyruvate mutase purified from T. pyriformis has been used to generate a precise oligonucleotide probe for the gene, using in vitro amplification from total genomic DNA by the polymerase chain reaction. Use of this precise probe and oligo(T) as primers for in vitro amplification from a T. pyriformis cDNA library has allowed the cloning of the mutase gene. A similar amplification strategy from genomic DNA yielded the genomic sequence, which contains three introns. The sequence of the DNA that encodes 10 amino acids upstream of the N-terminal sequence of the isolated protein was found by oligonucleotide hybridization to a subgenomic library. These 10 N-terminal amino acids are cleanly removed in Tetrahymena in vivo. The full mutase gene sequence codes for a protein of 300 amino acids, and it includes two amber (TAG) codons in the open reading frame. In Tetrahymena, TAG codes for glutamine. When the two amber codons are each changed to a glutamine codon (CAG) that is recognized by E. coli and the gene is placed behind a promoter driven by the T7 RNA polymerase, expression in E. coli is observed. The mutase gene also contains a large number of arginine AGA codons, a codon that is very rarely used by E. coli. Cotransformation with a plasmid carrying the dnaY gene [which encodes tRNA(Arg)(AGA)] results in more than 4-fold higher expression. The mutase then comprises about 25% of the total soluble cell protein in E. coli transformants. The mutase gene bears significant similarity to one other gene in the available data bases, that of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus, an enzyme that catalyzes a closely related transformation. Due to the large evolutionary distance between Tetrahymena and Streptomyces, this similarity can be interpreted as the first persuasive evidence that the biosynthesis of phosphonates is an ancient metabolic process.     

Effect of insulin on the incorporation of3H-inositol into the inositol phospholipids (PI,PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP₂) and GPIs. Treatment with 10^–5M insulin decreases the total³H-inositol incorporation and incorporation into PI. 24 h after 10^–6M insulin treatment there is an elevation of these parameters. Second treatment with 10^–6M insulin doubles and 10^–5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.