Microbial transformations of (−)-α- and (+)-β-thujone by fungi of Absidia species

The microbial transformations of (−)-α- and (+)-β-thujone (1a and 1b) in cultures of Absidia species: Absidia coerulea AM93, Absidia glauca AM254 and Absidia cylindrospora AM336 were studied. The biotransformations of (−)-α-thujone (1a), by these fungi strains, afforded mixtures of 4-hydroxy- and 7-hydroxy-α-thujone (2 and 3). Aforementioned fungi strains were also able to hydroxylate of (+)-β-thujone at C-7 position. Only A. glauca AM254 transformed 1b to 8-hydroxy-β-thujone (7) and (2S)-2-hydroxyneoisothujol (6). The (4R)-4-hydroxyisothujole (5) was identified as one of the major metabolite of (+)-β-thujone (1b) in culture of A. cylindrospora AM336. This strain was also able to introduce hydroxy group to C-4 position in 1b without reduction of carbonyl group at C-3. The absolute configuration of all chiral centers of new (4R)-4-hydroxyisothujol (5) and (2S)-2-hydroxyneoisothujol (6) were established taking into account the configuration of (+)-β-thujone (1b) and their spectral data.     

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3if-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.     

An embarrassment of sortases - a richness of substrates?

A range of surface proteins is anchored to the cell walls of Gram-positive pathogens such as Staphylococcus aureus by the transpeptidase sortase. Until now, sortase-like proteins and their substrates appeared to be limited mainly to such pathogens. However, by searching for sortase homologues among complete and incomplete genome sequences, we have found them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean. There is usually more than one sortase-like protein encoded in each Gram-positive genome, and the genes encoding the sortase-like proteins are often clustered with genes encoding their likely substrates.     

Biotransformation of (-)-(R)-α-Phellandrene: Antimicrobial Activity of Its Major Metabolite

Terpene derivatives converted by microbial biotransformation constitute an important resource for natural pharmaceutical, fragrance, and aroma substances. In the present study, the monoterpene α-phellandrene was biotransformed by 16 different strains of microorganisms (bacteria, fungi, and yeasts). The transformation metabolites were initially screened by TLC and GC/MS, and then further characterized by NMR spectroscopic techniques. Among the six metabolites characterized, 6-hydroxypiperitone, α-phellandrene epoxide, cis-p-menth-2-en-1-ol, and carvotanacetone, which originated from (-)-(R)-α-phellandrene, are reported for the first time in this study. Additionally, the substrate and the metabolite 5-p-menthene-1,2-diol were subjected to in vitro antibacterial and anticandidal tests. The metabolite showed moderate-to-good inhibitory activities (MICs=0.125 to >4?mg/ml) against various bacteria and especially against Candida species in comparison with its substrate (-)-(R)-α-phellandrene and standard antimicrobial agents.     

Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.     

Syntheses of p-aminophenyl 1-thio-α- and -β-d-idopyranosides as analogs of glycosidase substrates

The p-nitrophenyl and p-aminophenyl 1-thio-α- and -β-d-idopyranosides were synthesized for use in structure-activity studies of glycosidases. Zinc chloride-catalyzed fusion of α-d-idopyranose pentaacetate with p-nitrobenzenethiol gave p-nitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-α-d-idopyranoside as an amorphous glass in 67% yield, and the crystalline β anomer in 13% yield. Deacetylation with catalytic amounts of sodium methoxide in methanol, followed by hydrogenation under pressure over palladium-on-barium sulfate catalyst, afforded p-aminophenyl 1-thio-α- and -β-d-idopyranosides.     

Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.     

Synthesis and Cytotoxicity of О- and N-Acyl Derivatives of Azepanobetulin

Russian Journal of Bioorganic Chemistry - A five-stage synthesis of azepanobetulin from betulin with a total yield of 47% has been carried out. The acylation of azepanobetulin with anhydrides or...     

An acid-stable analogue of the 3-β-D-ribofuranoside of Y-base

A cyclonucleoside analogue of Y_TU riboside has been prepared and shown to be relatively stable in M-hydrochloric acid solution at room temperature.     

Unusual Role of the 3-OH Group of Oligosaccharide Substrates in the Mechanism of Bacillus 1,3-1,4-β-glucanase

Abstract

In the mechanism of retaining β-glycosidases, the 2-hydroxyl group of the substrate in the monosaccharyl unit involved in catalysis (subsite -1) is beleived to play an important role through hydrogen bonding interactions with protein residues that are optimized at the transition state. Commonly, removal of the 2-OH group of the substrate results in a 10–12 kcal·mol^-1 transition state destabilization. However, this effect seems not to be general as reported here for Bacillus 1,3-1,4-β-glucanase, a family 16 retaining endo-glycosidase. A p-nitrophenol 2-deosxy tetrasaccharide substrate was synthesized to probe the involvement of the 2-OH group in catalysis. Comparative kinetics with wild-type and subsite +1 mutants show that the 2-deoxy analog is a better substrate than the corresponding 2-hydroxy substrate. It is tentatively proposed that the 2-deoxy analog adopts a different conformation upon binding that compensates for the lack of the 2-OH substituent.     

Characterization of the α- and β-Mannosidases of Porphyromonas gingivalis

Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1→3 and α-1→2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1→6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated.     

Ribosylation of 3-Methylguanine and the Relative Stability of its 7- and 9-α-D-Ribofuranosides

Abstract

Ribosylation of 3-methylguanine la was investigated by enzymatic and chemical methods. Compound la did not act as a substrate for purine nucleoside phosphorylase. N-2-Protected 3-methylguanines 4 and 6 underwent exclusive N-7 glycosylation by fusion and chloromercury methods to give 5 and 7. Fully acetylated 7-α-D-ribofuranoside 5 was also obtained by thermal transglycosylation of the corresponding 9-α-D-ribofuranoside 9. The reverse isomerization 5 → 9 did not occur. The differences in the relative stability towards acidic hydrolysis between 7- and 9-(α-D-ribofuranosyl)-3-methylguanines are distinctly higher than those described so far for the other 7-9 isomeric nucleosides.     

Novel biocatalyst for productions of S-(-)-2-[6-benzyloxy -2,5,7,8-tetramethylchroman -2-yl] ethanol—precursor of natural α-tocols

A novel promising strain of actinobacteria Rhodococcus sp. 77-32 was identified. Its acetonetreated biomass the could be used as a biocatalyst for production of S-(-)-2-[6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl] ethanol (S-BCE), a precursor of natural α-tocols. It was established that a reaction of enantioselective hydrolysis of racemic (±)-2-(2-acetoxyethyl)-6-benzyloxy-2,5,7,8-tetramethylchroman (BCEA) occurred in the phosphate buffer–acetone system, resulting in enrichment of the residual substrate by S-enantiomer (S-(+)-2-(2-acetoxyethyl)-6-benzyloxy-2,5,7,8-tetramethylchroman, S-BCEA). It was shown that the hydrolysis was accompanied by stereoinversion of the formed product, R-(+)-2-[6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl] ethanol (R-BCE), into the S-BCE. The transformation conditions (acetone content, acidity, temperature, reaction duration) were optimized, providing simultaneous production of optically pure S-BCE and S-BCEA with an almost quantitative yield.     

Rational design of thermostability in bacterial 1,3-1,4-β-glucanases through spatial compartmentalization of mutational hotspots

Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures. The calcium region was predicted to be the crucial region for thermostability of bacterial 1,3-1,4-β-glucanases, and optimization of four residue sites in this region by iterative saturation mutagenesis greatly increased the thermostability of a mesophilic β-glucanase (BglT) from Bacillus terquilensis. The E46P/S43E/H205P/S40E mutant showed a 20 °C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature (T _m) compared to wild-type BglT. Its half-life values at 60 and 70 °C were 3.86-fold and 7.13-fold higher than those of wild-type BglT. The specific activity of E46P/S43E/H205P/S40E mutant was increased by 64.4 %, while its stability under acidic environment was improved. The rational design strategy used in this study might be applied to improve the thermostability of other industrial enzymes.

     

Synthesis and immunological evaluation of the 4-β-glucoside of HMBPP

HMBPP ((E)-4-hydroxy-3-methyl-2-butenyl pyrophosphate) is a highly potent innate immunogen that stimulates human γδ T cells expressing the Vγ2Vδ2 T cell antigen receptor. To determine if glycoside conjugates of HMBPP retain activity, the 4-β-glucoside and its acetylated homolog were synthesized and tested for their ability to stimulate γδ T cells. The glycoside HMBPP conjugate stimulated human γδ T cells with an EC(50) of 78nM. The tetraacetyl glycoside HMBPP conjugate was also active (EC(50)=360nM). The two isomeric mono-β-glucosides of the parent (E)-2-methylbut-2-ene-1,4-diol, however, were not active. Thus, HMBPP glycosylated at the 4-OH position stimulates γδ T cells as long as the pyrophosphate moiety is present.