Structural and functional analyses of human tryptophan 2,3‐dioxygenase

Tryptophan 2,3‐dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2‐C3 bond of l ‐tryptophan. This is a rate‐limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 Å resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in l ‐tryptophan oxidation. Proteins 2014; 82:3210–3216. © 2014 Wiley Periodicals, Inc.     

Conformations and properties of the L‐tryptophyl‐containing peptides in solution,depending on the pH—Theoretical study vs. experiments

The conformational preference and electronic properties of three L ‐tryptophyl‐containing dipeptides, i.e., glycyl‐L ‐tryptophane (H‐Gly‐Trp‐OH), L ‐alanyl‐L ‐tryptophane (H‐Ala‐Trp‐OH), and L ‐methionyl‐L ‐tryptophane (L ‐Met‐Trp‐OH) in solution depending on the pH of the media are studied both theoretically and experimentally. The effect of the protonation of the COO^? and deprotonation of the NH as well as the alkaline hydrolysis of the amide fragment in a strong basic media on the electronic spectra are discussed. Ab initio and density functional theory (DFT) methods as well as the time‐dependent DFT (TD‐DFT) method as a function of the basis set are performed with a view to obtain the geometry and electronic properties of all of the species as well as the intermediate, obtained in the alkaline hydrolysis mechanism. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 727–734, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Purified silk fibroin‐L‐asparaginase bioconjugates show increased thermostability and resistance to trypsin digestion

Bioconjugation protocols have been recently used to improve the therapeutic properties of the anti‐leukemic enzyme L ‐asparaginase. In this study, we study the variation of reaction factors, such as mass ratio, pH value, reaction temperature and time, and the concentration of cross‐linker, in the modification reaction of L ‐asparaginase with silk fibroins. The optimum reaction parameters were obtained as follows: pH 7.0, mass ratio of silk fibroin to L ‐asparaginase 5:1, reaction time 8 h, and temperature 4°C. The rate of ε‐amino group modification was 57.88% and the recovery of modified L ‐asparaginase was 66.58% under these reaction conditions. We isolated and purified the silk‐L ‐asparaginase conjugates with two consecutive chromatography steps: anion‐exchange (XK 16×20, Q Sepharose FF) and gel filtration (Tricorn 10×600, Sephcryl S‐300 HR) chromatography. Finally, the stabilities of the enzymes were investigated. The results showed that modified L ‐asparaginase had a higher thermostability and higher resistance to trypsin digestion.     

Asymmetrically simultaneous synthesis of L‐homophenylalanine and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L ‐Homophenylalanine (L ‐HPA) and N⁶‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N⁶‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N⁶‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N⁶‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N⁶‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Diffusion of sucrose and yohimbine in calcium alginate gel beads with or without entrapped plant cells

The diffusion characteristics of sucrose, a nutrient, and yohimbine, a secondary metabolite, in alginate gel beads, with or without entrapped periwinkle (Catharanthus roseus) or apple (Malus domestica) cells, were investigated. Effective diffusivities of both solutes in the gel beads were determined by two different methods from transient concentration changes in well-stirred solutions where the beads were suspended. The linear plot method developed in this work is easy to use and requires no data from the initial periods of diffusion experiments. It was found that while the cell-free beads provided only minor diffusional resistance to both solutes, the effective diffusivities of both solutes decreased significantly with the presence of cells in the beads and the amount of reduction was proportional to the amount of cell loading. Further, the effective diffusivity of sucrose appeared to be slightly larger than that of yohimbine under identical conditions. It was also observed that permeabilization of apple cells with dimethyl sulfoxide (DMSO) led to an increase in effective diffusivity with the effect being more significant for yohimbine.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Simultaneous synthesis of 2‐phenylethanol and L‐homophenylalanine using aromatic transaminase with yeast Ehrlich pathway

2‐Phenylethanol is a widely used aroma compound with rose‐like fragrance and L ‐homophenylalanine is a building block of angiotensin‐converting enzyme (ACE) inhibitor. 2‐phenylethanol and L ‐homophenylalanine were synthesized simultaneously with high yield from 2‐oxo‐4‐phenylbutyric acid and L ‐phenylalanine, respectively. A recombinant Escherichia coli harboring a coupled reaction pathway comprising of aromatic transaminase, phenylpyruvate decarboxylase, carbonyl reductase, and glucose dehydrogenase (GDH) was constructed. In the coupled reaction pathway, the transaminase reaction was coupled with the Ehrlich pathway of yeast; (1) a phenylpyruvate decarboxylase (YDR380W) as the enzyme to generate the substrate for the carbonyl reductase from phenylpyruvate (i.e., byproduct of the transaminase reaction) and to shift the reaction equilibrium of the transaminase reaction, and (2) a carbonyl reductase (YGL157W) to produce the 2‐phenylethanol. Selecting the right carbonyl reductase showing the highest activity on phenylacetaldehyde with narrow substrate specificity was the key to success of the constructing the coupling reaction. In addition, NADPH regeneration was achieved by incorporating the GDH from Bacillus subtilis in the coupled reaction pathway. Based on 40 mM of L ‐phenylalanine used, about 96% final product conversion yield of 2‐phenylethanol was achieved using the recombinant E. coli. Biotechnol. Bioeng. 2009;102: 1323–1329. © 2008 Wiley Periodicals, Inc.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Diffusion in kappa-carrageenan gel beads

Using a well-mixed and temperature-led vessel, the diffusion characteristics of various solutes into spherical kappa-carrageenan gel beads were experimentally investigated. The diffusion coefficient of glucose was markedly affected by the glucose concentration and the operating temperature. In all cases the diffusivity obtained was noticeably smaller than that of glucose in pure water. The experimental data also indicated an inverse relationship between the diffusivity and the polymer concentration used in the gel preparation. As well, the glucose diffusivity was affected by the presence of other solutes in the glucose solution. Electrolytes such as ammonium sulfate, KCl, and CaCl(2) were observed to enhance the diffusion coefficient. On the other hand, the addition of arginine or bovine serum albumin had an adverse effect on the diffusivity. No diffusion of albumin into the gel beads was observed, and such a solute created a significant mass transfer resistance during the diffusion process.     

High‐level conversion of L‐lysine into 5‐aminovalerate that can be used for nylon 6,5 synthesis

L ‐Lysine is a potential feedstock for the production of bio‐based precursors for engineering plastics. In this study, we developed a microbial process for high‐level conversion of L ‐lysine into 5‐aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta‐aminovaleramidase (DavA) and lysine 2‐monooxygenase (DavB) was grown to high density in fed‐batch culture and used as a whole cell catalyst. High‐density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD₆₀₀) of 30, yielded 36.51 g/L 5AVA from 60 g/L L ‐lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L ‐lysine in 24 h. 5AVA production was further improved by doubling the L ‐lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L ‐lysine by E. coli WL3110 cells grown to OD₆₀₀ of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ?‐caprolactam and δ‐valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio‐nylon production processes.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

MicroRNA‐93‐5p expression in tumor tissue and its tumor suppressor function via targeting programmed death ligand‐1 in colorectal cancer

This work aimed to investigate miR‐93‐5p expression in tumor tissue and its in vitro effects in colorectal cancer (CRC) by targeting programmed death ligand‐1 (PD‐L1). MiR‐93‐5p and PD‐L1 expression was detected in CRC and adjacent normal tissues by quantitative real‐time polymerase chain reaction and immunohistochemistry. The correlation between miR‐93‐5p and PD‐L1 was validated by a dual‐luciferase reporter assay. HCT116 and SW480 cells were divided into blank, miR‐NC, miR‐93‐5p mimics, miR‐93‐5p inhibitor, PD‐L1 small interfering RNA (siRNA) and miR‐93‐5p inhibitor + PD‐L1 siRNA groups, and wound‐healing and transwell assays were performed to detect cell migration and invasion, respectively. Protein expression was measured by western blotting. The secretion of cytokines was detected in the CRC cell/T coculture models. MiR‐93‐5p was downregulated in CRC tissues with upregulated PD‐L1. In PD‐L1‐negative patients, miR‐93‐5p expression was increased compared with that in PD‐L1‐positive patients. MiR‐93‐5p and PD‐L1 expression levels were associated with the tumor differentiation, lymphatic metastasis, TNM, Duke's stage, and prognosis of CRC. PD‐L1 siRNA weakened the migration and invasion abilities via decreased expression of matrix metalloproteinase‐1 (MMP‐1), ‐2, and ‐9, and these effects were abolished by the miR‐93‐5p inhibitor. Additionally, anti‐PD‐L1 upregulated the expressions of interleukin‐2 (IL‐2), tumor necrosis factor‐α (TNF‐α), and interferon γ (IFN‐γ) in the coculture of T cells with CRC cells, but downregulated the expressions of IL‐1β, IL‐10, and TGF‐β. However, these changes were partially reversed by miR‐93‐5p inhibition. miR‐93‐5p is expected to be a novel target for CRC treatment since it decreases the migration and invasion, as well as the immune evasion, of CRC cells via targeting PD‐L1.     

Oxidative DNA Damage Induced by a Copper(II)?1,10‐Phenanthroline?L‐Serine Complex in the Presence of Rutin

The capacity of the ternary complex copper(II)? 1,10‐phenanthroline? L ‐serine ([Cu? Phen? Ser]) to induce double‐strand scission of DNA was explored by agarose‐gel electrophoresis. It was found that the complex exhibited remarkable activity to damage DNA in the presence of rutin. Analysis of the UV and fluorescence spectra clearly demonstrated that the complex was bound to DNA by intercalation. Further, the occurrence of 8‐hydroxydeoxyguanosine (8‐OHdG), a biomarker of oxidative DNA damage, after the treatment of DNA by the complex in presence of rutin was evidenced by an electrochemical method. Finally, the mechanism of oxidative damage to double‐stranded DNA by the [Cu? Phen? Ser] complex in the presence of rutin was discussed.     

Monte Carlo simulation of 4‐α‐glucanotransferase reaction

4‐α‐Glucanotransferase (GTase, D ‐enzyme) catalyzes disproportionation between two short polymers of maltooligosaccharides linked by α‐1,4‐glucoside bonds. Using action modes of the potato GTase for the donor and acceptor substrates, the Monte Carlo method was applied to simulate the GTase reaction. The simulation starts from a single enzyme molecule and a finite number (10⁵) of substrate molecules. All selection processes were performed using random numbers produced by computer. The initial substrates were from trimer to 10‐mer. In every case, the final stage was the steady‐state distribution of polymers. The steady‐state distribution by the potato GTase reaction was different from those by the hypothetical random disproportionation reaction. The simulated data from the reaction of potato GTase and trimer almost quantitatively agreed with experimental data. The mechanism of the GTase reaction was accumulation of probabilistic processes and was well simulated by the Monte Carlo method. GTase randomizes the overall distribution of chain length of the substrate. Therefore the GTase reaction is an entropy‐driven process. © 1999 John Wiley & Sons, Inc. Biopoly 50: 145–151, 1999     

Partitioning of amino acids in the aqueous biphasic system containing the water‐miscible ionic liquid 1‐butyl‐3‐methylimidazolium bromide and the water‐structuring salt potassium citrate

In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio‐molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)—a new class of non‐volatile alternative solvents. ABS composed of the hydrophilic IL {1‐butyl‐3‐methylimidazolium bromide ([C₄mim]Br)} and potassium citrate—which is easily degraded—represents a clean media to green separation of bio‐molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL ‐based ABS, the distribution of five model amino acids (L ‐tryptophan, L ‐phenylalanine, L ‐tyrosine, L ‐leucine, and L ‐valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting‐out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL ‐based ABS, a model was established to describe the partition coefficient of three model amino acids, L ‐tryptophan, L ‐phenylalanine, and L ‐valine, and employed to predict the partition coefficient of two other model amino acids, L ‐tyrosine and L ‐leucine. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Enantioselective Synthesis and Antimicrobial Activities of Tetrahydro‐Β‐Carboline Diketopiperazines

A series of single isomers tetrahydro‐β‐carboline diketopiperazines were stereoselectively synthesized starting from l ‐tryptophan methyl ester hydrochloride and six aldehydes through a four‐step reaction including Pictet‐Spengler reaction, crystallization‐induced asymmetric transformations (CIAT), Schotten‐Baumann reaction, and intramolecular ester amidation. The chemical structures were characterized by nuclear magnetic resonance (NMR) and elemental analysis, among which two compounds were determined by x‐ray single crystal diffraction. Moreover, antimicrobial activities of all the compounds were also tested. Chirality 25:656–662, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibiting the color formation by gradient temperature‐elevating Maillard reaction of soybean peptide‐xylose system based on interaction of l‐cysteine and Amadori compounds

Light color and savory flavor enhancer are attractive for consumers and food producers. The effect of addition time of l ‐cysteine on inhibiting color formation was investigated in soybean peptide‐xylose system, and the possible pathway was explored. Once dicarbonyl compounds were formed during the Maillard reaction, the addition of l ‐cysteine had no color‐inhibiting effect; if l ‐cysteine was added immediately after the Amadori compound was formed, the extraordinary color‐inhibiting effect was observed. Therefore, an improved way to inhibit color formation was proposed on the basis of the interaction of l ‐cysteine and Amadori compounds by controlling the addition time of l ‐cysteine through gradient temperature‐elevating Maillard reaction. The system was heated at 80 °C for 60 min to form Amadori compounds, followed by the addition of L‐cysteine, and the temperature was raised to 120 °C and held for 110 min. Compared with traditional products, the lightest color product was found desirable by GC/MS analysis and sensory evaluation. The novel method proposed can be a guide for the industrial preparation of light‐colored products. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Z‐FA.FMK activates duodenal epithelial cell proliferation through oxidative stress,NF‐κB and IL‐1β in d‐GalN/TNF‐α‐administered mice

This study was designed to evaluate the effect of Z‐FA.FMK (benzyloxycarbonyl‐l ‐phenylalanyl‐alanine‐fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z‐FA.FMK, the third group received d ‐GalN (d ‐galactosamine) and TNF‐α (tumour necrosis factor‐α) and the fourth group was given both d ‐GalN/TNF‐α and Z‐FA.FMK. When d ‐GalN/TNF‐α was administered alone, we observed an increase in IL‐1β‐positive and active NF‐κB‐positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)‐positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z‐FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d ‐GalN/TNF‐α injection. On the contrary, the Z‐FA.FMK pretreatment before d ‐GalN/TNF‐α blocked these effects. Based on these findings, we suggest that Z‐FA.FMK might act as a proliferative mediator which is controlled by IL‐1β through NF‐κB and oxidative stress in duodenal epithelial cells of d ‐GalN/TNF‐α‐administered mice.