Dependence of crystallographic atomic displacement parameters on temperature (25-150†K) for complexes of horse liver alcohol dehydrogenase.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Šresolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Šand is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Catalytic asymmetric Tsuji-Trost α-benzylation reaction of N-unprotected amino acids and benzyl alcohol derivatives.

Catalytic asymmetric Tsuji-Trost benzylation is a promising strategy for the preparation of chiral benzylic compounds. However, only a few such transformations with both good yields and enantioselectivities have been achieved since this reaction was first reported in 1992, and its use in current organic synthesis is restricted. In this work, we use N-unprotected amino acid esters as nucleophiles in reactions with benzyl alcohol derivatives. A ternary catalyst comprising a chiral aldehyde, a palladium species, and a Lewis acid is used to promote the reaction. Both mono- and polycyclic benzyl alcohols are excellent benzylation reagents. Various unnatural optically active α-benzyl amino acids are produced in good-to-excellent yields and with good-to-excellent enantioselectivities. This catalytic asymmetric method is used for the formal synthesis of two somatostatin mimetics and the proposed structure of natural product hypoestestatin 1. A mechanism that plausibly explains the stereoselective control is proposed.

Elucidation of the Transport Mechanism of Puerarin and Gastrodin and Their Interaction on the Absorption in a Caco-2 Cell Monolayer Model.

Puerarin (PUR) and gastrodin (GAS) are often used in combined way for treating diseases caused by microcirculation disorders. The current study aimed to investigate the absorption and transportation mechanism of PUR and GAS and their interaction via Caco-2 monolayer cell model. In this work, the concentration in Caco-2 cell of PUR and GAS was determined by HPLC method. The bidirectional transport of PUR and GAS and the inhibition of drug efflux including verapamil and cyclosporine on the transport of these two components were studied. The mutual influence between PUR and GAS, especially the effect of the latter on the former of the bidirectional transport were also investigated. The transport of 50 µg·mL-1 PUR in Caco-2 cells has no obvious directionality. While the transport of 100 and 200 µg·mL-1 PUR presents a strong directionality, and this directionality can be inhibited by verapamil and cyclosporine. When PUR and GAS were used in combination, GAS could increase the absorption of PUR while PUR had no obvious influence on GAS. Therefore, the compatibility of PUR and GAS is reasonable, and GAS can promote the transmembrane transport of PUR, the effect of which is similar to that of verapamil.

Structure of dye-decolorizing peroxidase from Bacillus subtilis in complex with veratryl alcohol.

Dye-decolorizing peroxidases (DyPs) are heme-containing peroxidases, which have promising application in biodegradation of phenolic lignin compounds and in detoxification of dyes. In this study, the crystal structure of BsDyP- veratryl alcohol (VA) complex delves deep into the binding of small substrate molecules within the DyP heme cavity. The biochemical analysis shows that BsDyP oxidizes the VA with a turnover number of 0.065 s-1, followed by the oxidation of 2,6-dimethoxyphenol (DMP) and guaiacol with a comparable turnover number (kcat) of 0.07 s-1 and 0.07 s-1, respectively. Moreover, biophysical and computational studies reveal the comparable binding affinity of substrates to BsDyP and produce lower-energy stable BsDyP-ligand(s) complexes. All together with our previous findings, we are providing a complete structural description of substrate-binding sites in DyP. The structural insight of BsDyP helps to modulate its engineering to enhance the activity towards the oxidation of a wide range of substrates.

Insight into the protein salting-in mechanism of arginine, magnesium chloride and ethylene glycol: Solvent interaction with aromatic solutes.

Key factors in the salting-in effects on proteins of additives are their interactions with aromatic groups. We studied the interaction of four aromatic solutes, benzyl alcohol (BA), phenol, 4-hydroxybenzyl alcohol (4-HBA) and methyl gallate (MG), with different salting-in additives, arginine hydrochloride (ArgHCl), magnesium chloride (MgCl2), ethylene glycol (EG), and guanidine hydrochloride (GdnHCl) using solubility measurements. We used sodium chloride (NaCl) as a control. MgCl2 decreased the solubility of the four aromatic solutes with weak solute dependence. In contrast, ArgHCl, GdnHCl, and EG increased the solubility of four aromatic solutes with a similar solute dependence. Their salting-in effects were weaker on BA and 4-HBA and stronger on phenol and MG. These results indicate that attached groups alter the aromatic properties, affecting the interactions between the benzene ring and these three additives. More importantly, the observed results demonstrate that the salting-in mechanism is different between MgCl2, EG and ArgHCl, which should play a role in their effects on protein solubility.

Gentisyl alcohol and homogentisic acid: Plasmodium falciparum dihydroorotate dehydrogenase inhibitors isolated from fungi.

Two Indonesian fungi Aspergillus assiutensis BioMCC-f.T.7495 and Penicillium pedernalense BioMCC-f.T.5350 along with a Japanese fungus Hypomyces pseudocorticiicola FKI-9008 have been found to produce gentisyl alcohol (1), which inhibits Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with an IC50 value of 3.4 µM. Another Indonesian fungus, Penicillium citrinum BioMCC-f.T.6730, produced an analog of 1, homogentisic acid (4), which also inhibits PfDHODH with an IC50 value of 47.6 µM.

The Infuence of Salicin on Rheological and Film-Forming Properties of Collagen.

Collagen films are widely used as adhesives in medicine and cosmetology. However, its properties require modification. In this work, the influence of salicin on the properties of collagen solution and films was studied. Collagen was extracted from silver carp skin. The rheological properties of collagen solutions with and without salicin were characterized by steady shear tests. Thin collagen films were prepared by solvent evaporation. The structure of films was researched using infrared spectroscopy. The surface properties of films were investigated using Atomic Force Microscopy (AFM). Mechanical properties were measured as well. It was found that the addition of salicin modified the roughness of collagen films and their mechanical and rheological properties. The above-mentioned parameters are very important in potential applications of collagen films containing salicin.

Mechanistic study of the biosynthesis of R-phenylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations.

The biosynthesis of R-phenylacetylcarbinol (R-PAC) by the acetohydroxy acid synthase, (AHAS) is addressed by molecular dynamics simulations (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and QM/MM free energy calculations. The results show the reaction starts with the nucleophilic attack of the C2α atom of the HEThDP intermediate on the Cß atom of the carbonyl group of benzaldehyde substrate via the formation of a transition state (TS1) with the HEThDP intermediate under 4'-aminopyrimidium (APH+) form. The calculated activation free energy for this step is 17.4kcal mol-1 at 27 °C. From this point, the reaction continues with the abstraction of Hß atom of the HEThDP intermediate by the Oß atom of benzaldehyde to form the intermediate I. The reaction is completed with the cleavage of the bond C2α-C2 to form the product R-PAC and to regenerate the ylide intermediate under the APH+ form, allowing in this way to reinitiate to the catalytic cycle once more. The calculated activation barrier for this last step is 15.9kcal mol-1 at 27 °C.

H2O2-Responsive Antioxidant Nanoparticle Attenuates Whole Body Ischemia/Reperfusion-Induced Multi-Organ Damages.

Mortality and morbidity after cardiac arrest remain high due to ischemia/reperfusion (I/R) injury causing multi-organ damages, even after successful return of spontaneous circulation. We previously generated H2O2-activatable antioxidant nanoparticles formulated with copolyoxalate containing vanillyl alcohol (PVAX) to prevent I/R injury. In this study, we examined whether PVAX could effectively reduce organ damages in a rat model of whole-body ischemia/reperfusion injury (WBIR). To induce a cardiac arrest, 70µl/100 g body weight of 1 mmol/l potassium chloride was administered via the jugular venous catheter. The animals in both the vehicle and PVAX-treated groups had similar baseline blood pressure. After 5.5 minutes of cardiac arrest, animals were resuscitated via intravenous epinephrine followed by chest compressions. PVAX or vehicle was injected after the spontaneous recovery of blood pressure was noted, followed by the same dose of second injection 10 minutes later. After 24 hours, multiple organs were harvested for pathological, biochemical, molecular analyses. No significant difference on the restoration of spontaneous circulation was observed between vehicle and PVAX groups. Analysis of organs harvested 24 hours post procedure showed that whole body I/R significantly increased reactive oxygen species (ROS) generation, inflammatory markers, and apoptosis in multiple organs (heart, brain, and kidney). PVAX treatment effectively blocked ROS generation, reduced the elevation of pro-inflammatory cytokines, and decreased apoptosis in these organs. Taken together, our results suggest that PVAX has potent protective effect against WBIR induced multi-organ injury, possibly by blocking ROS-mediated cell damage.

Antioxidant and Antibacterial Activity of Caprylic Acid Vanillyl Ester Produced by Lipase-Mediated Transesterification.

Vanillyl alcohol (VA), which is abundant in Vanilla bean, has strong antioxidant activity. However, the use of VA in the food and cosmetics industries is limited, due to its low solubility in emulsion or organic solvents. Meanwhile, medium chain fatty acids and medium chain monoglycerides have antibacterial activity. We synthesized butyric acid vanillyl ester (BAVE) or caprylic acid vanillyl ester (CAVE) from VA with tributyrin or tricaprylin through transesterification reaction using immobilized lipases. BAVE and CAVE scavenged 2,2-diphenyl-1-picrylhydrazyl radicals in organic solvents. In addition, BAVE and CAVE decreased the production rate of conjugated diene and triene in the menhaden oil-in-water emulsion system. While BAVE showed no antibacterial activity, CAVE showed antibacterial activity against food spoilage bacteria, including Bacillus coagulans. In this study, the antibacterial activity of vanillyl ester with medium chain fatty acid was first revealed. Zeta potential measurements confirmed that BAVE and CAVE were inserted into B. coagulans membrane. In addition, the propidium iodide uptake assay and fluorescent microscopy showed that CAVE increased B. coagulans membrane permeability. Therefore, CAVE is expected to play an important role in the food and cosmetics industries as a bi-functional material with both antioxidant and antibacterial activities.

Selective hydrodeoxygenation of lignin model compound (3,4-dimethoxybenzyl alcohol) by Pd/CNX catalyst.

Upgrading of lignin derived bio-oil is an essential step for producing sustainable bio-based chemicals and fuel. Taken into account that α hydroxyl is the abundant functional group in lignin, high effective and selective catalytic alcoholysis for cleaving the Cα-OH linkages would be desirable. However, an in-depth understanding of the reaction mechanisms involved in the cleavage of Caromatic-Cα and Cα-O bonds over a novel catalyst is still needed. Herein, we report an efficient liquid-phase hydrogen transfer strategy for the selective hydrodeoxygenation of a non-phenolic lignin model compound, 3,4-dimethoxybenzyl (veratryl) alcohol, under mild conditions. By employing iso-propanol as solvent and H-donor, and palladium nanoparticles immobilized on nitrogen-doped carbon (Pd/CNX) as efficient multifunctional catalyst, veratryl alcohol dehydroxylation exhibited almost 100% conversion along with very high selectivity for 1,2-dimethoxy-benzene (46%) and 3,4-dimethoxytoluene (54%). Compared with other Pd catalysis, the Pd/CNX has excellent catalytic performances and exhibits higher selectivity for 3,4-dimethoxytoluene under incorporation with 1% HCOOH at 220 °C. The proportion of Pd (0) significantly increases in Pd/CNX catalyst when introduced into N precursor because of its highly dispersed Pd NPs and preventing the reoxidation of Pd (0). The dehydrogenation reaction occurred through the hydrogen generation of a secondary alcohol. Then, the Cα-OH and Caromatic-Cα bonds of veratryl alcohol were selectively cleaved by catalytic transfer hydrogenolysis. The alcoholysis mechanism is supported by dispersion-corrected density functional theory computations.

Enzymatic decolorization of melanin by lignin peroxidase from Phanerochaete chrysosporium.

Skin darkening results as a consequence of the accumulation of skin pigment melanin. To combat this, the amplitude of skin lightening agents are commercially available, most of which inhibit melanin synthesis. Decolorization of melanin is an alternative method of skin lightening. In this study, we show that lignin peroxidase (LiP), an extracellular enzyme purified from Phanerochaete chrysosporium NK-1 isolated from a forest soil can effectively degrade and decolorize melanin in vitro. Decolorization conditions including pH, temperature, incubation time, enzyme concentration, and mediator addition were investigated to optimize the reaction conditions. The results indicate that pH 3, 40 °C, 15 IU/ml, and 10 h incubation were the optimal conditions for the decolorization of the melanin. The use of the mediator, veratryl alcohol was also found effective to enhance the efficacy of the melanin decolonization, with up to 92% decolorization. The scanning electron microscopy results showed void spaces on the treated melanin granules as compared to the untreated sample, indicating the degradation of melanin. Changes in the fingerprint region of the melanin were observed. Between wavenumbers 1500-500 cm-1, for example, the presence of new peaks in the treated melanin at 1513, 1464, and 1139 cm-1 CH2, CH3 bend and C-O-C stretch represented structural changes. A new peak at 2144 cm-1 (alkynyl C≡C stretch) was also detected in the decolorized melanin. The cytotoxicity study has shown that the treated melanin and LiP have low cytotoxic effects; however, the mediator of veratryl alcohol could result in high mortality which suggests that its use should be meticulously tested in formulating health and skincare products. The findings of the study suggest that LiP produced by Phanerochaete chrysosporium has the potential to be used in the medical and cosmetic industries, particularly for the development of biobased cosmetic whitening agents.

A novel diclofenac-hydrogel conjugate system for intraarticular sustained release: Development of 2-pyridylamino-substituted 1-phenylethanol (PAPE) and its derivatives as tunable traceless linkers.

A local sustained-release drug delivery system, or depot, for intra-articular injection offers the opportunity to release a therapeutic agent directly to the joint with limited need for reinjection. A successful system would provide more consistent efficacy and minimize systemic side effects. In this paper, we explore the potential use of diclofenac, a non-steroidal anti-inflammatory drug, for use in a polymer-conjugate depot system. During the course of our exploration it was determined that "conventional ester" conjugates of diclofenac were not appropriate as upon incubation in buffer (pH 7.4) or in bovine synovial fluid, a considerable amount of undesired diclofenac-lactam was released. Thus we developed a novel linker system for diclofenac in order to minimize the production of the lactam. This new linker enables a diclofenac conjugate system with tunable release rates and minimizes the production of undesired lactam side-products.

Tissue-Adhesive Paint of Silk Microparticles for Articular Surface Cartilage Regeneration.

Current biomaterials and tissue engineering techniques have shown a promising efficacy on full-thickness articular cartilage defect repair in clinical practice. However, due to the difficulty of implanting biomaterials or tissue engineering constructs into a partial-thickness cartilage defect, it remains a challenge to provide a satisfactory cure in joint surface regeneration in the early and middle stages of osteoarthritis. In this study, we focused on a ready-to-use tissue-adhesive joint surface paint (JS-Paint) capable of promoting and enhancing articular surface cartilage regeneration. The JS-Paint is mainly composed of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide (NB)-coated silk fibroin microparticles and possess optimal cell adhesion, migration, and proliferation properties. NB-modified silk fibroin microparticles can directly adhere to the cartilage and form a smooth layer on the surface via the photogenerated aldehyde group of NB reacting with the -NH2 groups of the cartilage tissue. JS-Paint treatment showed a significant promotion of cartilage regeneration and restored the smooth joint surface at 6 weeks postsurgery in a rabbit model of a partial-thickness cartilage defect. These findings revealed that silk fibroin can be utilized to bring about a tissue-adhesive paint. Thus, the JS-Paint strategy has some great potential to enhance joint surface regeneration and revolutionize future therapeutics of early and middle stages of osteoarthritis joint ailments.

The improvement effect of gastrodin on LPS/GalN-induced fulminant hepatitis via inhibiting inflammation and apoptosis and restoring autophagy.

Fulminant hepatitis (FH), characterized by overwhelmed inflammation and massive hepatocyte apoptosis, is a life-threatening and high mortality rate. Gastrodin (GTD), a phenolic glucoside extracted from Gastrodiaelata Blume, exerts anti-apoptosis, and anti-inflammatory activities. In the present study, we aimed to evaluate whether GTD treatment could alleviate lipopolysaccharide and d-galactosamine (LPS/GalN)-induced FH in mice and its potential mechanisms. These data suggested that GTD treatment remarkably protected against LPS/GalN-induced FH by enhancing the survival rate of mice, reducing ALT and AST levels, attenuating histopathological changes, and suppressing interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α secretion. In addition, GTD treatment relieved hepatic apoptosis by the regulation of peroxisome proliferator-activated receptors (PPARs), P53 and caspase-3/9. Furthermore, GTD treatment could significantly inhibit inflammation-related signaling pathways activated by LPS/GalN, including the suppression of nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) and nuclear factor-kappa B (NF-κB) activation. Importantly, GTD treatment effectively restored but not induced LPS/GalN-reduced the expression of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, as well as the level of pro-autophagy proteins. Taken together, our investigation indicated that GTD played an essential role in liver protection by relieving hepatocyte apoptosis and inflammation reaction, which may be closely involved in the inhibition of NLRP3 inflammasome and NF-κB activation, regulation of apoptosis-related proteins expression, and the recovery of AMPK/ACC/autophagy.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

Miyabeacin: A new cyclodimer presents a potential role for willow in cancer therapy.

Willow (Salix spp.) is well known as a source of medicinal compounds, the most famous being salicin, the progenitor of aspirin. Here we describe the isolation, structure determination, and anti-cancer activity of a cyclodimeric salicinoid (miyabeacin) from S. miyabeana and S. dasyclados. We also show that the capability to produce such dimers is a heritable trait and how variation in structures of natural miyabeacin analogues is derived via cross-over Diels-Alder reactions from pools of ortho-quinol precursors. These transient ortho-quinols have a role in the, as yet uncharacterised, biosynthetic pathways around salicortin, the major salicinoid of many willow genotypes.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Elastomeric polyurethane porous film functionalized with gastrodin for peripheral nerve regeneration.

The extracellular matrix provides cells with a support structure and an attachment site in actual substrate. Its biochemical and surface properties play an important role in and have significant impact on cell attachment, proliferation, migration, differentiation, and gene expression. Leveraging the hydrophilicity and neuroprotective of gastrodin, a gastrodin/polyurethane (PU) elastomer was developed utilizing in situ polymerization and salt-leaching methods. The results showed that gastrodin/PU film had a good flexibility and supporting strength, as well as hydrophilicity. Thus film possessed highly surface area, interconnected porous structure with a pore size (10~60 µm) for cell attachment, and could provide surface cues to augment neurite extension. For PC12 cells cultured within the films, especially the 5gastrodin/PU group, presented a progressive increase with time, coupled with the upregulation of brain-derived neurotrophic factor and glial cell derived neurotrophic factor expression. This is the first report on the construction of a gastrodin/PU porous film, and the results reveal its promise as a scaffold material for neural tissue engineering.

Special Issue: Development of Asymmetric Synthesis.

Biological systems usually respond differently to enantiomers of a chiral molecule due to the inherent chirality of the active receptor sites of enzymes in nature [...].