Engineering the promiscuous racemase activity of an arylmalonate decarboxylase

Variant G74C of arylmalonate decarboxylase (AMDase) from Bordatella bronchoseptica has a unique racemising activity towards profens. By protein engineering, variant G74C/V43A with a 20-fold shift towards promiscuous racemisation was obtained, based on a reduced activity in the decarboxylation reaction and a two-fold increase in the racemisation activity. The mutant showed an extended substrate range, with a 30-fold increase in the reaction rate towards ketoprofen. Molecular dynamics simulations and the substrate profile of the racemase indicate that the steric and polar effects of the substrate structure play a more dominant role on catalysis than mere kinetic α-proton acidity. The observation that the conversion of β,γ-unsaturated carboxylic acids does not lead to a rearrangement to form their α,β isomers indicates a concerted rather than a stepwise mechanism. Interestingly, a substrate bearing a nitro group instead of the carboxylic acid group on the α-carbon atom was also converted by the racemase.     

Heat-treated magnesia as an adsorbent for the class separation of aliphatic monocarbonyl compounds

In order to control the activity of magnesia as an adsorbent, the effect of temperature was studied. The activity of various temperature of treatment showed two regions. During the first, in the range 106–320°, the activity increased with increasing temperature, whereas at temperatures above 320° the activity decreased with increasing temperature.The iodine number of heat-treated magnesia correlated well with its affinity for 2,4-dinitrophenylhydrazones of carbonyls.By heating several batches of magnesia at graphically determined temperatures. it was possible to obtain adsorbents with the same activity for class separation.     

Effect of the degrees of polymerization of an enzyme and a substrate on the catalytic activity of the enzyme

It is shown that, as the degree of polymerization of an enzyme molecule is decreased, its enzymatic activity declines and subsequently vanishes at the triple point of the state diagram of a macromolecule. As the degree of polymerization of the enzyme molecule is increased, its enzymatic activity becomes higher owing to an increase in the density of loops in the fringelike surface layer of the globule. An expression for the turnover number of the enzyme that allows for effects of the chemical structures of the enzyme and the substrate on enzymatic activity is obtained.     

Towards the understanding of the activity of G9a inhibitors: an activity landscape and molecular modeling approach

In this work, we analyze the structure–activity relationships (SAR) of epigenetic inhibitors (lysine mimetics) against lysine methyltransferase (G9a or EHMT2) using a combined activity landscape, molecular docking and molecular dynamics approach. The study was based on a set of 251 G9a inhibitors with reported experimental activity. The activity landscape analysis rapidly led to the identification of activity cliffs, scaffolds hops and other active an inactive molecules with distinct SAR. Structure-based analysis of activity cliffs, scaffold hops and other selected active and inactive G9a inhibitors by means of docking followed by molecular dynamics simulations led to the identification of interactions with key residues involved in activity against G9a, for instance with ASP 1083, LEU 1086, ASP 1088, TYR 1154 and PHE 1158. The outcome of this work is expected to further advance the development of G9a inhibitors.

     

CmlI is an N-oxygenase in the biosynthesis of chloramphenicol

The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated in Escherichia coli using two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC–MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.     

Dihydrobenzofurans as cannabinoid receptor ligands from Cordyceps annullata,an entomopathogenic fungus cultivated in the presence of an HDAC inhibitor

The secondary metabolite production of Cordyceps annullata, an entomopathogenic fungus, was clearly enhanced by the addition of suberoyl bis-hydroxamic acid (SBHA), a histone deacetylase (HDAC) inhibitor, to the culture medium. Four new 2,3-dihydrobenzofurans, annullatins A–D (1–4), and a new aromatic polyketide, annullatin E (5) were isolated from the culture medium, and the structures, including the absolute stereochemistries, were determined by spectroscopic analysis, vibrational circular dichroism (VCD), and chemical transformations. Annullatin A (1) exhibited potent agonistic activity toward the cannabinoid receptors CB1 and CB2. Annullatin B (2) and D (4) displayed CB1 agonistic activity and CB2 inverse agonistic activity.     

Using Toad Oocytes as an Ultrasensitive System for Estimating the Biological Activity of Trichosanthin

A sensitive bioassay for estimating the activity of Trichosanthin was developed by using intraoocyte microinjection as the technique and germinal vescicle breakdown as a marker. The GVBD inhibitory activity assay was million-fold more sensitive than the routinely used abortifacient activity assay. The detectable amount of Trichosanthin ranged from 0.5 to 100 pg. Since the least detectable dose was 0.5 pg, it made the assay suitable to analyse the microquantity of pure Trichosanthin used for studying the structure and function of the drug.     

Effect of catalysts on the reaction of an aliphatic isocyanate and water

The kinetics of the reaction of aliphatic isocyanate with water were investigated with hexyl isocyanate as a model compound. The kinetic study was carried out with a titration method to determine the concentration of the isocyanate group as a function of time. Gas chromatography was used to augment the kinetic data obtained from the titration method. The effects of an organic acid [p‐toluene sulfonic acid monohydrate (p‐TSA)], a tertiary amine {diazabicyclo[2.2.2]octane (DABCO)}, and an organotin compound [dibutyltin dilaurate (DBTDL)] on the reaction were investigated for the conversion of isocyanate to a urea. Under the reaction conditions in this study, urea was the only product observed. The rate constants indicated that p‐TSA had low catalytic activity, DABCO had intermediate catalytic activity, and DBTDL had high catalytic activity. A reaction mechanism was proposed for each of the catalysts. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1677–1688, 2002     

Activating an enzyme by an engineered coiled coil switch

We designed a de novo protein based on a circular permutant of RNaseT1, in which the enzymatic activity can be manipulated by engineered peptide binding. The circular permutant of RNaseT1 was obtained by tethering the original C- and N-termini with a GPAG linker and cleaving the molecule between Glu82 and Asn83. This mutant lacked enzymatic activity, due to the destabilization of entire protein structure. We previously reported the construction of ABC-type heterotrimeric coiled coil peptides, in which the A- and B-type peptides cannot form the folded trimeric structure without the C-type peptide. The introduction of the A- and B-type coiled coil peptides to the C- and N-termini of the circular permutant of RNaseT1, respectively, and the subsequent addition of the C-type coiled coil peptide enabled the RNaseT1 domain to refold properly, thus, restoring the enzymatic activity. The formation of the trimeric coiled coil structure should bring the cleaved sites of RNaseT1 close enough to refold the RNaseT1 domain spontaneously.     

Complanine, an inflammation-inducing substance isolated from the marine fireworm Eurythoe complanata

The marine fireworm, Eurythoe complanata, is known as a dangerous animal for humans because it induces skin inflammation through its small setae. Here, the inflammation-inducing substance was successfully isolated from the whole body using a methanolic extraction, and the inflammatory activity was determined using a bioassay. The structure was spectroscopically revealed to be a trimethylammonium with an unsaturated carbon chain and was named complanine. Complanine enhanced PKC activity in combination with TPA in vitro. This may explain the molecular mechanism behind its inflammation-inducing activity.     

Effect of the addition of D-xylose on xylanase activity and digestibility of fiber in an artificial rumen

The effect of adding D-xylose at two different levels as a continuous infusion to an artificial rumen was determined with respect to pH, methane production, volatile fatty acid production, fiber digestion, and xylanase activity. No changes in pH, methane production, or fiber digestion were observed as consequences of increased D-xylose concentrations. Although the propionic acid production was slightly raised on addition of D-xylose, the acetic acid production was lowered to the same extent. A low-xylose infusion increased the xylanase activity by 25%, whereas a high-xylose infusion lowered the activity by 17%. It was concluded that D-xylanase activity was not a rate-determining step in fiber degradation in the rumen.     

Cyclopalladated ferrocenylimine as an efficient catalyst for the syntheses of diarylmethane derivatives

The cyclopalladated ferrocenylimine adducts Ia , Ib , Ic were evaluated in the Suzuki cross‐coupling reaction of benzyl halides with arylboronic acids. The tricyclohexylphosphine adduct Ia exhibited highly catalytic activity for the coupling of aryl and heteroaryl boronic acids containing various functional groups with benzylic bromides and chlorides (up to 99% yield), furnishing diarylmethane derivatives with low catalyst loading (1 mol%). It is worth noting that catalyst Ia can be reused eight times without losing its catalytic activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Consideration of an activity of the metallocene catalyst by using molecular mechanics,molecular dynamics and QSAR

Recently metallocene based catalyst are widely used for the olefin polymerization catalyst. It is well known however that the metallocene catalysts do not show any activity without cocatalyst and also their activity depends on content and species of cocatalyst. In this study, the most stable structures of various catalyst–cocatalyst systems were calculated by using molecular mechanics (MM) and molecular dynamics (MD). Various structural parameters which are related to the space between central atoms of catalyst and cocatalyst, charges, and interaction energy were obtained from those structures. By using the quantitative activity–structure relationship (QSAR) analysis, the space between catalyst and cocatalyst was found to be the main factor to control the activity. By using this acknowledgment the activity of the metallocene catalyst may be roughly estimated without using the heavy ab initio molecular orbital calculation.     

Upregulation of an Artificial Zymogen by Proteolysis

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.     

Nanostructured platinum as an electrocatalyst for the electrooxidation of formic acid

Nanostructured platinum prepared by the chemical reduction of hexachloroplatinic acid dissolved in aqueous domains of the liquid crystalline phases of oligoethylene oxide surfactants, was examined as an electrocatalyst for the electrooxidation of formic acid. The electrocatalytic properties of the catalyst combining highly specific surface areas and a periodic mesoporous nanostructure were accessed in sulfuric acid solution containing 0.5 mol dm⁻³ formic acid using cyclic voltammetry (CV) and chronoamperometry. The electrocatalytic activity of the material at 60 °C, is characterised by a mass activity of 8.6 A g⁻¹ and a specific surface area activity of 26 μA cm⁻² at 0.376 V (vs. RHE). The resistance to CO poisoning was found to depend upon electrode potential. At hydrogen adsorption potentials, the material is easily poisoned, while the material shows high resistance to CO poisoning at potentials positive of the hydrogen region. These facts suggest that the decomposition of HCOOH on the mesoporous platinum is likely to proceed through a dual-path mechanism and the high surface area material is a potential electocatalyst towards the electrooxidation of small organic molecules.     

Synthesis and an evaluation of the bioactivity of the C-glycoside of pseudopterosin A methyl ether

The Suzuki-Miyaura cross-coupling protocol was applied to the synthesis of 1a, the C-glycoside analogue of PsA methyl ether. This marks the first construction of a C-glycoside for this class of marine natural products, thereby offering an opportunity to compare its bioactivity to the natural substances. Its activity profile resembled that of PsA (1) and PsA O-methyl ether (1b) when assayed for its anti-inflammatory activity and its ability to inhibit phagocytosis. We conclude that the intact structure is present when a pseudopterosin expresses its anti-inflammatory and phagocytosis inhibitory properties and that they are, therefore, not likely to be prodrugs. Results show that 1a is an effective binding agent toward the A2A and A3 adenosine receptors, displaying IC50 values of 20 and 10 microM, respectively.     

Discovery of an orally-active nonsteroidal androgen receptor pure antagonist and the structure-activity relationships of its derivatives

The 3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethylthiohydantoin derivatives which have carboxy-terminal side chains were synthesized and their agonistic/antagonistic activities against androgen receptor (AR) measured. Among them, compound 13b showed antagonistic activity (IC50=130 nM) with no agonistic activity even at 10000 nM. This compound exhibited significant metabolic stability and oral antiandrogenic activity (ED50=7 mg/kg).     

Activity of an enzyme immobilized on polyelectrolyte multilayers

The immobilization of α-chymotrypsin on the surface of boron silicate glass microspheres is conducted via the technique of multilayer adsorption of polyelectrolytes. It is shown that the enzyme is adsorbed on both positively and negatively charged surfaces and its activity is partially preserved relative to that in solution. The activity of the enzyme depends on the number of polyectrolyte layers preliminarily adsorbed on glass microspheres and on the charge of the surface. The activity of α-chymotrypsin adsorbed on the negatively charged surface is four times higher than the activity of this enzyme adsorbed on a positively charged surface.     

Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model

The chemical composition and antibacterial activity of essential oils from 10 commonly consumed herbs: Citrus aurantium, C. limon, Lavandula angustifolia, Matricaria chamomilla, Mentha piperita, M. spicata, Ocimum basilicum, Origanum vulgare, Thymus vulgaris and Salvia officinalis have been determined. The antibacterial activity of these oils and their main components; i.e. camphor, carvacrol, 1,8-cineole, linalool, linalyl acetate, limonene, menthol, a-pinene, b-pinene, and thymol were assayed against the human pathogenic bacteria Bacillus subtilis, Enterobacter cloacae, Escherichia coli O157:H7, Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, S. epidermidis, S. typhimurium, and Staphylococcus aureus. The highest and broadest activity was shown by O. vulgare oil. Carvacrol had the highest antibacterial activity among the tested components.     

Towards an identification of the pyrethroid pharmacophore. A molecular modelling study of some pyrethroid esters

Summary A molecular modelling and computer graphics study of a series of pyrethroid insecticides has been carried out. The three-dimensional arrangement of the groups essential for the biological activity (pharmacophore) has been identified for the acid and the alcohol moieties, respectively. These pharmacophores are based on the relationship between molecular structure and biological activity for a number of pyrethroid esters. The pharmacophores, which describe the relative location in space of the unsaturated systems, the dimethyl groups and the ester moiety, may be useful in the design of novel compounds with pyrethroid activity.