Molecular dynamics simulations of active site mutants of triosephosphate isomerase

Molecular dynamics simulations of triosephosphate isomerase(TIM) and of some active site TIM mutants were performed inan attempt to elucidate possible interactions important forcatalytic activity and binding. A variety of active site residuesin TIM have been altered, resulting in all cases in decreasesin catalytic activity. Second-site suppressor mutants were characterizedfor two of these active site mutants. The pseudorevertants haveincreased activity compared to the single mutant from whichthey were derived and, surprisingly, in both cases the increasehi activity is a result of the replacement of an active siteserine for proline. We performed simulations of wild-type TIMand the active site mutants with the substrate dihydroxyacetonephosphate bound both non-covalently and covalently. The noncovalentcomplexes were used to examine interactions important to bindingwhile the covalent complexes are models of the transition statestructure for enolization, which is the rate-determining stepfor the mutants. The difference between these two states, then,is related to the catalytic activity. We found various protein-substrateinteractions that unproved in the noncovalent mutant complexes,which correlates with the experimentally observed increase inbinding affinity upon mutation. In the covalent complexes weobserved improved electrostatic stabilization of the transitionstate upon introduction of Pro, which is also consistent withthe experimental data. Our simulations reproduce the highlyco-operative nature of the interactions in the active site andsuggest that this approach may be useful for identifying particularlypromising sites for mutation.     

Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase

The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a verytight and rigid dimer interface. At this interface a criticalhydrogen bond is formed between the main chain oxygen atom ofthe catalytic residue Lys13 and the completely buried side chainof Gln65 (of the same subunit). The sequence of Leishmania mexicanaTIM, closely related to Trypanosoma brucei TIM (68% sequenceidentity), shows that this highly conserved glutamine has beenreplaced by a glutamate. Therefore, the 1.8 Å crystalstructure of leishmania TIM (at pH 5.9) was determined. Thecomparison with the structure of trypanosomal TIM shows no rearrangementsin the vicinity of Glu65, suggesting that its side chain isprotonated and is hydrogen bonded to the main chain oxygen ofLys13. Ionization of this glutamic acid side chain causes apH-dependent decrease in the thermal stability of leishmaniaTIM. The presence of this glutamate, also in its protonatedstate, disrupts to some extent the conserved hydrogen bond network,as seen in all other TIMs. Restoration of the hydrogen bondingnetwork by its mutation to glutamine in the E65Q variant ofleishmania TIM results in much higher stability; for example,at pH 7, the apparent melting temperature increases by 26°C(57°C for leishmania TIM to 83°C for the E65Q variant).This mutation does not affect the kinetic properties, showingthat even point mutations can convert a mesophilic enzyme intoa superstable enzyme without losing catalytic power at the mesophilictemperature.     

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.     

Inhibition of Enzyme Activity of Rhipicephalus (Boophilus) microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.     

Optimization of baker's yeast drying in industrial continuous fluidized bed dryer

Instant active dry baker's yeast is a well-known product widely used for leavening of bread, produced by fermentation, and usually dried by hot air to 94-96% dry matter content. Multi-stage fluidized bed drying process is a commercial effective method for yeast drying. In this work, optimum operating parameters of an industrial continuous fluidized bed dryer for the production of instant active dry yeast were investigated. The dryer contained four zones separated with moving weirs. The operating conditions such as temperature, loading rate of compressed yeast granules, and hot air humidity had direct effects on both yeast activity and viability. The most important factors that affected the quality of the product were loading rate and the operational temperature in each zone on the bed. Optimization was performed for three loading rates of the feed to the dryer, using response surface methodology for the experimental design. The most significant factor was shown to be the loading rate with mean fermentation activity values of 620, 652, and 646 cm³ CO₂/h for 300, 350, and 400 kg/h loading rates, respectively. The data analysis resulted in an optimal operating point at a loading rate of 350 kg/h and temperatures of zones 1, 2, 3, and 4 controlled at 33, 31, 31, and 29 °C, respectively. The best activity value was predicted as 668 ± 18 cm³ CO₂/h, and confirmation experiments resulted in 660 ± 10 cm³ CO₂/h. At the same operating point, the average viability of the cells was predicted as 74.8 ± 3.7% and confirmed as 76.4 ± 0.6%. Compared with the normal operating conditions at the plant, the optimization resulted in more than 12% and 27% improvement in the yeast activity and viability, respectively.     

Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.     

Effects of Low Molecular Weight Yeast β-Glucan on Antioxidant and Immunological Activities in Mice

To evaluate the antioxidant and immune effects of low molecular yeast β-glucan on mice, three sulfated glucans from Saccharomyces cerevisiae (sGSCs) with different molecular weight (M_W) and degrees of sulfation (DS) were prepared. The structures of the sGSCs were analyzed through high performance liquid chromatography-gel permeation chromatography (HPLC-GPC) and Fourier transform infrared spectroscopy (FTIR). sGSC1, sGSC2, and sGSC3 had M_W of 12.9, 16.5 and 19.2 kDa, respectively, and DS of 0.16, 0.24 and 0.27, respectively. In vitro and in vivo experiments were conducted to evaluate the antioxidant and immunological activities of the sGSCs. In vitro experiment, the reactive oxygen species (ROS) scavenging activities were determined. In vivo experiment, 50 male BALB/c mice were divided into five groups. The sGSC1, sGSC2 and sGSC3 treatment groups received the corresponding sGSCs at 50 mg/kg/day each. The GSC (glucans from Saccharomyces cerevisiae) treatment group received 50 mg/kg/day GSC. The normal control group received equal volume of physiological saline solution. All treatments were administered intragastrically for 14 day. Results showed that sGSC1, sGSC2 and sGSC3 can scavenge 1,1-diphenyl-2-picryl-hydrazyl (DPPH), superoxide, and hydroxyl radicals in vitro. The strength of the radical scavenging effects of the sGSCs was in the order of sGSC1 > sGSC2 > sGSC3. Oral administration of sGSC1 significantly improved serum catalase (CAT) and glutathione peroxidase (GSH-Px) activities and decreased malondialdehyde (MDA) level in mice. sGSC1 significantly improved the spleen and thymus indexes and the lymphocyte proliferation, effectively enhanced the percentage of CD4⁺ T cells, decreased the percentage of CD8⁺ T cells, and elevated the CD4⁺/CD8⁺ ratio. sGSC1 significantly promoted the secretion of IL-2 and IFN-γ. These results indicate that sGSC1 with low M_W and DS has better antioxidant and immunological activities than the other sGSCs, and sGSC1 could be used as a new antioxidant and immune-enhancing agent.     

Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.     

Different Reactive Oxygen Species Lead to Distinct Changes of Cellular Metal Ions in the Eukaryotic Model Organism Saccharomyces cerevisiae

Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al(3+)) level rose up to 50-fold after the diamide treatment. Cellular potassium (K(+)) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al(3+) accumulation was further validated by the enhanced Al(3+) uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al(3+) uptake, suggesting Al(3+)-specific transporters could be involved in Al(3+) uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.     

The Effect of C-Terminal Helix on the Stability of FF Domain Studied by Molecular Dynamics Simulation

To investigate the effect of C-terminal helix on the stability of the FF domain, we studied the native domain FF3-71 from human HYPA/FBP11 and the truncated version FF3-60 with C-terminal helix being deleted by molecular dynamics simulations with GROMACS package and GROMOS 43A1 force field. The results indicated that the structures of truncated version FF3-60 were evident different from those of native partner FF3-71. Compared with FF3-71, the FF3-60 lost some native contacts and exhibited some similar structural characters to those of intermediate state. The C-terminal helix played a major role in stabilizing the FF3-71 domain. To a certain degree, the FF domain had a tendency to form an intermediate state without the C-terminal helix. In our knowledge, this was the first study to examine the role of C-terminal helix of FF domain in detail by molecular dynamics simulations, which was useful to understand the three-state folding mechanism of the small FF domain.     

煅烧对硫酸钙晶须结构及稳定性的影响

不同温度下煅烧硫酸钙晶须,并用XRD测定煅烧产物的结构,发现其晶型和晶格结构均有变化,600 ℃左右煅烧的硫酸钙晶须晶格结构最为致密.硫酸钙晶须的水化试验表明:不同温度煅烧的硫酸钙晶须具有不同的稳定性, 600 ℃以上温度煅烧的硫酸钙晶须具有很好的稳定性,并且煅烧硫酸钙晶须的晶型和晶格结构差异是其稳定性不同的主要原因.     

Biofuel Production Based on Carbohydrates from Both Brown and Red Macroalgae: Recent Developments in Key Biotechnologies

Marine macroalgae (green, red and brown macroalgae) have attracted attention as an alternative source of renewable biomass for producing both fuels and chemicals due to their high content of suitable carbohydrates and to their advantages over terrestrial biomass. However, except for green macroalgae, which contain relatively easily-fermentable glucans as their major carbohydrates, practical utilization of red and brown macroalgae has been regarded as difficult due to the major carbohydrates (alginate and mannitol of brown macroalgae and 3,6-anhydro-l-galactose of red macroalgae) not being easily fermentable. Recently, several key biotechnologies using microbes have been developed enabling utilization of these brown and red macroalgal carbohydrates as carbon sources for the production of fuels (ethanol). In this review, we focus on these recent developments with emphasis on microbiological biotechnologies.     

Engineering the substrate specificity of xylose isomerase

Xylose isomerase (XI) catalyzes the isomerization and epimerization of hexoses, pentoses and tetroses. In order to clarify the reasons for the low reaction efficiency of a pentose sugar, L-arabinose, we determined the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. The crystal structure revealed that, when compared with D-xylose and D-glucose, L-arabinose binds to the active site in a partially different position, in which the ligand has difficulties in binding the catalytic metal M2. Lys183 has been thought to stabilize the open substrate conformation by hydrogen bonding to oxygen O1. Our results with L-arabinose showed that the substrate stays in a linear form even without a hydrogen bond between Lys183 and oxygen O1. We engineered mutations to the active site of Actinoplanes missouriensis XI to improve the reaction efficiency with L-arabinose. The mutation F26W was intended to shift the position of oxygen O1 of L-arabinose closer to the catalytic metal M2. This effect of F26W was modeled by free energy perturbation simulations. In line with this, F26W increased 2-fold the catalytic efficiency of XI with L-arabinose; the increase was seen mainly in kcat. The mutation Q256D was outside the sphere of the catalytic residues and probably modified the electrostatic properties of the active site. It improved 3-fold the catalytic efficiency of XI with L-arabinose; this increase was seen in both Km and kcat. This study showed that it is possible to engineer the substrate specificity of XI.     

Alternating current electrophoretic deposition of Saccharomyces cerevisiae cells and the viability of the deposited biofilm in ethanol production

In this work, we have explored the possibility of using asymmetrical alternating current electrophoretic deposition (AC-EPD) process, which we have previously reported for enzyme deposition, to immobilize Saccharomyces cerevisiae (S. cerevisiae) cells onto stainless steel substrates. The deposition of S. cerevisiae cells at 30 Hz and 200 V_p-p permits the formation of 89 ± 16 μm thick cell layers in 30 min. The mass of the deposited cells is shown to increase quasi-linearly with the deposition time and the applied amplitude. In order to increase the mechanical stability of the immobilized cells, a thin layer of polyurethane was applied after the AC-EPD of S. cerevisiae cells. The viability of the immobilized cells was tested in the production of ethanol. The results showed that the fermentation process with the immobilized S. cerevisiae cells is more efficient than the fermentation carried out with similarly treated free cells.     

Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15

Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties.     

Carotenoids, Fatty Acid composition and heat stability of supercritical carbon dioxide-extracted-oleoresins

The risk of chronic diseases has been shown to be inversely related to tomato intake and the lycopene levels in serum and tissue. Cis-isomers represent approximately 50%-80% of serum lycopene, while dietary lycopene maintains the isomeric ratio present in the plant sources with about 95% of all-trans-lycopene. Supercritical CO(2) extraction (S-CO(2)) has been extensively developed to extract lycopene from tomato and tomato processing wastes, for food or pharmaceutical industries, also by using additional plant sources as co-matrices. We compared two S-CO(2)-extracted oleoresins (from tomato and tomato/hazelnut matrices), which showed an oil-solid bi-phasic appearance, a higher cis-lycopene content, and enhanced antioxidant ability compared with the traditional solvent extracts. Heat-treating, in the range of 60-100 °C, led to changes in the lycopene isomeric composition and to enhanced antioxidant activity in both types of oleoresins. The greater stability has been related to peculiar lycopene isomer composition and to the lipid environment. The results indicate these oleoresins are a good source of potentially healthful lycopene.     

The Structure and Dynamics of BmR1 Protein from Brugia malayi: In Silico Approaches

Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in “Brugia Rapid”. However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.     

Potential Activity of Fevicordin-A from Phaleria macrocarpa (Scheff) Boerl. Seeds as Estrogen Receptor Antagonist Based on Cytotoxicity and Molecular Modelling Studies

Fevicordin-A (FevA) isolated from Phaleria macrocarpa (Scheff) Boerl. seeds was evaluated for its potential anticancer activity by in vitro and in silico approaches. Cytotoxicity studies indicated that FevA was selective against cell lines of human breast adenocarcinoma (MCF-7) with an IC₅₀ value of 6.4 μM. At 11.2 μM, FevA resulted in 76.8% cell death of T-47D human breast cancer cell lines. Critical pharmacophore features amongst human Estrogen Receptor-α (hERα) antagonists were conserved in FevA with regard to a hypothesis that they could make notable contributions to its pharmacological activity. The binding stability as well as the dynamic behavior of FevA towards the hERα receptor in agonist and antagonist binding sites were probed using molecular dynamics (MD) simulation approach. Analysis of MD simulation suggested that the tail of FevA was accountable for the repulsion of the C-terminal of Helix-11 (H11) in both agonist and antagonist receptor forms. The flexibility of loop-534 indicated the ability to disrupt the hydrogen bond zipper network between H3 and H11 in hERα. In addition, MM/GBSA calculation from the molecular dynamic simulations also revealed a stronger binding affinity of FevA in antagonistic action as compared to that of agonistic action. Collectively, both the experimental and computational results indicated that FevA has potential as a candidate for an anticancer agent, which is worth promoting for further preclinical evaluation.     

Bioethanol production from corn meal by simultaneous enzymatic saccharification and fermentation with immobilized cells of Saccharomyces cerevisiae var. ellipsoideus

The simultaneous enzymatic saccharification and fermentation (SSF) of corn meal using immobilized cells of Saccharomycescerevisiae var. ellipsoideus yeast in a batch system was studied. The yeast cells were immobilized in Ca-alginate by electrostatic droplet generation method. The process kinetics was assessed and determined and the effect of addition of various yeast activators (mineral salts: ZnSO₄ · 7H₂O and MgSO₄ · 7H₂O, and vitamins: Ca-pantothenate, biotin and myo-inositol) separately or mixed, was investigated. Taking into account high values of process parameters (such as ethanol concentration, ethanol yield, percentage of the theoretical ethanol yield, volumetric productivity and utilized glucose) and significant energy savings the SSF process was found to be superior compared to the SHF process. Further improvement in ethanol production was accomplished with the addition of mineral salts as yeast activators which contributed to the highest increase in ethanol production. In this case, the ethanol concentration of 10.23% (w/w), percentage of the theoretical ethanol yield of 98.08%, the ethanol yield of 0.55 g/g and the volumetric productivity of 2.13 g/l·h were obtained.