- Direct monitoring of biocatalytic deacetylation of amino acid substrates by1H NMR reveals fine details of substrate specificity
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Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that thel-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range ofN-acyl-amino acid substrates. This activity was revealed by1H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.
- De Cesare, Silvia,McKenna, Catherine A.,Mulholland, Nicholas,Murray, Lorna,Bella, Juraj,Campopiano, Dominic J.
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supporting information
p. 4904 - 4909
(2021/06/16)
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- Argicyclamides A-C Unveil Enzymatic Basis for Guanidine Bis-prenylation
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Guanidine prenylation is an outstanding modification in alkaloid and peptide biosynthesis, but its enzymatic basis has remained elusive. We report the isolation of argicyclamides, a new class of cyanobactins with unique mono- and bis-prenylations on guanidine moieties, from Microcystis aeruginosa NIES-88. The genetic basis of argicyclamide biosynthesis was established by the heterologous expression and in vitro characterization of biosynthetic enzymes including AgcF, a new guanidine prenyltransferase. This study provides important insight into the biosynthesis of prenylated guanidines and offers a new toolkit for peptide modification.
- Balloo, Nandani,Fujita, Kei,Matsuda, Kenichi,Okino, Tatsufumi,Phan, Chin-Soon,Wakimoto, Toshiyuki
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supporting information
p. 10083 - 10087
(2021/07/26)
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- Sequence-Selective Protection of Peptides from Proteolysis
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Proteolysis of proteins and peptides is involved in the infection of cells by enveloped viruses and also in the invasion and spread of cancer cells. Shutting down broad-specificity proteases, however, is problematic because normal functions by these proteases will be affected. Herein, nanoparticle receptors were prepared from molecular imprinting for complex biological peptides. Their strong and selective binding enabled them to protect their targeted sequences from proteolysis in aqueous solution at stoichiometric amounts. Generality of the method was demonstrated by the protection of hydrophobic and hydrophilic peptides from different proteases, selective protection of a segment of a long peptide, and selective protection of a targeted peptide in a mixture. Most interestingly, two receptors targeting different parts of a long peptide could work in cooperation to protect the overall sequence, highlighting the versatility of the method.
- Li, Xiaowei,Chen, Kaiqian,Zhao, Yan
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supporting information
p. 11092 - 11097
(2021/04/05)
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- Binding Methylarginines and Methyllysines as Free Amino Acids: A Comparative Study of Multiple Host Classes**
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Methylated free amino acids are an important class of targets for host-guest chemistry that have recognition properties distinct from those of methylated peptides and proteins. We present comparative binding studies for three different host classes that are each studied with multiple methylated arginines and lysines to determine fundamental structure-function relationships. The hosts studied are all anionic and include three calixarenes, two acyclic cucurbiturils, and two other cleft-like hosts, a clip and a tweezer. We determined the binding association constants for a panel of methylated amino acids using indicator displacement assays. The acyclic cucurbiturils display stronger binding to the methylated amino acids, and some unique patterns of selectivity. The two other cleft-like hosts follow two different trends, shallow host (clip) following similar trends to the calixarenes, and the other more closed host (tweezer) binding certain less-methylated amino acids stronger than their methylated counterparts. Molecular modelling sheds some light on the different preferences of the various hosts. The results identify hosts with new selectivities and with affinities in a range that could be useful for biomedical applications. The overall selectivity patterns are explained by a common framework that considers the geometry, depth of binding pockets, and functional group participation across all host classes.
- Bayer, Peter,Hof, Fraser,Isaacs, Lyle,Kamba, Bianca E.,Le, My-Hue,Schrader, Thomas,Warmerdam, Zoey
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- Mutations of key substrate binding residues of leishmanial peptidase T alter its functional and structural dynamics
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Background: M20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues. Methods: PepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods. Results: PepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P1 position. Peptidomimetics amastatin and actinonin occupied S1 pocket by competing with the substrate for binding to active site and inhibited PepT potently, while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme. Conclusions: PepT preferentially hydrolyzes oligopeptides carrying alanine at P1 position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions. General significance: This study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.
- Bhat, Saleem Yousuf,Qureshi, Insaf Ahmed
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- Development of a Raltegravir-based Photoaffinity-Labeled Probe for Human Immunodeficiency Virus-1 Integrase Capture
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Photoaffinity labeling (PAL) is one of the upcoming and powerful tools in the field of molecular recognition. It includes the determination of dynamic parameters, such as the identification and localization of the target protein and the site of drug binding. In this study, a photoaffinity-labeled probe for full-length human immunodeficiency virus-1 integrase (HIV-1 IN) capture was designed and synthesized, following the structure of the FDA-approved drug Raltegravir. This photoprobe was found to retain the HIV IN inhibitory potential in comparison with its parent molecule and demonstrates the ability to label the HIV-1 IN protein. Putative photoprobe/inhibitor binding sites near the catalytic site were then identified after protein digestion coupled to mass and molecular modeling analyses.
- Pala, Nicolino,Esposito, Francesca,Tramontano, Enzo,Singh, Pankaj Kumar,Sanna, Vanna,Carcelli, Mauro,Haigh, Lisa D.,Satta, Sandro,Sechi, Mario
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supporting information
p. 1986 - 1992
(2020/11/09)
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- Lipophilic Arginine Esters: The Gateway to Preservatives without Side Effects
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This study hypothesized that long carbon chain cationic arginine (Arg) esters can be considered as toxicologically harmless preservatives. Arg-esters with C18 and C24 carbon chains, namely, arginine-oleate (Arg-OL) and arginine-decyltetradecanoate (Arg-DT), were synthesized. Structures were confirmed by FT-IR, 1H NMR, and mass spectroscopy. Both Arg-esters were tested regarding hydrophobicity in terms of log Poctanol/water, critical micelle concentration (CMC), biodegradability, cytotoxicity, hemolysis, and antimicrobial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), and Enterococcus faecalis (E. faecalis). Log Poctanol/water of arginine was raised from -1.9 to 0.3 and 0.6 due to the attachment of C18 and C24 carbon chains, respectively. The critical micelle concentration of Arg-OL and Arg-DT was 0.52 and 0.013 mM, respectively. Both Arg-esters were biodegradable by porcine pancreatic lipase. In comparison to the well-established antimicrobials, benzalkonium chloride (BAC) and cetrimide, Arg-esters showed significantly less cytotoxic and hemolytic activity. Both esters exhibited pronounced antimicrobial properties against Gram-positive and Gram-negative bacteria comparable to that of BAC and cetrimide. The minimum inhibitory concentration (MIC) of Arg-esters was a high potential of Arg-esters with long carbon chains as toxicologically harmless novel preservatives.
- Asim, Mulazim Hussain,Gust, Ronald,Hupfauf, Andrea,Jalil, Aamir,Knabl, Ludwig,Nelles, Philipp Alexander,Shahzadi, Iram,Bernkop-Schnürch, Andreas
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p. 3129 - 3139
(2020/09/16)
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- Role of metal cations and oxyanions in the regulation of protein arginine phosphatase activity of YwlE from Bacillus subtilis
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Protein arginine phosphorylation (pArg) is a relatively novel posttranslational modification. Protein arginine phosphatase YwlE negatively regulates arginine phosphorylation and consequently induces the expression of stress-response genes that are crucial for bacterial stress tolerance and pathogenic homolog Staphylococcus aureus virulence. However, little is known about the factors that affect the enzymatic activity of YwlE with the exception of the effect of oxidative stress. Herein, based on the hydrolysis of the chromogenic substrate p-nitrophenyl phosphate (pNPP) by YwlE, we investigate the role of metal cations and oxyanions in the regulation of YwlE activity. Interestingly, among the various cations that we tested, Ca2+ activates YwlE, while other cations, including Ag+, Co2+, Cd2+, and Zn2+, are inhibitory. Furthermore, as chemical analogues of phosphate, oxyanions play multiple roles in phosphatase activity. The regulatory switch Cys within the catalytic site regulates YwlE activity. Specifically, the thiol of this Cys could be alkylated by IAM (iodoacetamide) or oxidized by H2O2, resulting in enzymatic inhibition. Conversely, reducing reagents, such as DTT (dithiothreitol), β-me (β-mercaptoethanol), and TCEP (tris(2-carboxyethyl)phosphine) enhance YwlE activity. Additionally, as a stable analogue to pArg, pAIE binds to YwlE with a Kd of 149.1 nM and a binding stoichiometry n of 1.2 and inhibits YwlE with an IC50 of 316.3 ± 12.73 μM. The inhibition and activation of YwlE may have broad implications for the physiology, pharmacology and toxicology of metal cations and oxyanions.
- Huang, Biling,Huang, Chenyang,Huang, Shaohua,Liao, Xinli,Liu, Yan,Zhang, Yumeng,Zhao, Mingxiao,Zhao, Yufen,Zhao, Zhixing
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- Preparation and characterization of a new open-tubular capillary column for enantioseparation by capillary electrochromatography
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In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open-tubular (OT) column, in this study, a new OT-CEC, coated with cationic cyclodextrin (1-allylimidazolium-β-cyclodextrin [AI-β-CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI-β-CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI-β-CD-coated column were optimized with the orthogonal experiment design L9(34). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI-β-CD-coated columns was about 0.2 to 0.5?μm. The AI-β-CD content in stationary phase based on the EA was approximately 2.77?mmol·m?2. The AI-β-CD-coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI-β-CD, has a great potential for enantioseparation in OT-CEC.
- Li, Yingjie,Tang, Yimin,Qin, Shili,Li, Xue,Dai, Qiang,Gao, Lidi
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p. 283 - 292
(2019/02/05)
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- Bottom-Up Construction of an Adaptive Enzymatic Reaction Network
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The reproduction of emergent behaviors in nature using reaction networks is an important objective in synthetic biology and systems chemistry. Herein, the first experimental realization of an enzymatic reaction network capable of an adaptive response is reported. The design is based on the dual activity of trypsin, which activates chymotrypsin while at the same time generating a fluorescent output from a fluorogenic substrate. Once activated, chymotrypsin counteracts the trypsin output by competing for the fluorogenic substrate and producing a non-fluorescent output. It is demonstrated that this network produces a transient fluorescent output under out-of-equilibrium conditions while the input signal persists. Importantly, in agreement with mathematical simulations, we show that optimization of the pulse-like response is an inherent trade-off between maximum amplitude and lowest residual fluorescence.
- Helwig, Britta,van Sluijs, Bob,Pogodaev, Aleksandr A.,Postma, Sjoerd G. J.,Huck, Wilhelm T. S.
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supporting information
p. 14065 - 14069
(2018/10/09)
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- Chromatographic Resolution of α-Amino Acids by (R)-(3,3'-Halogen Substituted-1,1'-binaphthyl)-20-crown-6 Stationary Phase in HPLC
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Three new chiral stationary phases (CSPs) for high-performance liquid chromatography were prepared from R-(3,3'-halogen substituted-1,1'-binaphthyl)-20-crown-6 (halogen = Cl, Br and I). The experimental results showed that R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 (CSP-1) possesses more prominent enantioselectivity than the two other halogen-substituted crown ether derivatives. All twenty-one α-amino acids have different degrees of separation on R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6-based CSP-1 at room temperature. The enantioselectivity of CSP-1 is also better than those of some commercial R-(1,1'-binaphthyl)-20-crown-6 derivatives. Both the separation factors (α) and the resolution (Rs) are better than those of commercial crown ether-based CSPs [CROWNPAK CR(+) from Daicel] under the same conditions for asparagine, threonine, proline, arginine, serine, histidine and valine, which cannot be separated by commercial CR(+). This study proves the commercial usefulness of the R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 chiral stationary phase.
- Wu, Peng,Wu, Yuping,Zhang, Junhui,Lu, Zhenyu,Zhang, Mei,Chen, Xuexian,Yuan, Liming
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supporting information
p. 1037 - 1042
(2017/07/25)
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- Purification, structural characterization and bioactivity evaluation of a novel proteoglycan produced by Corbicula fluminea
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A novel proteoglycan, named CFPS-11, was isolated from Corbicula fluminea, which is a food source of freshwater bivalve mollusk. CFPS-11 had an average molecular weight of 807.7 kDa and consisted of D-glucose and D-glucosamine in a molar ratio of 12.2:1.0. The protein moiety (～5%) of CFPS-11 was covalently bonded to the polysaccharide chain in O-linkage type through both serine and thereonine residues. The polysaccharide chain of CFPS-11 was composed of (1 → 4)-α-D-glucopyranosyl and (1 → 3,6)-α-D-glucopyranosyl residues, which branched at O-6. The branch chain consisted of (1 →)-α-D-glucopyranosyl and (1 →)-α-D-N-acetylglucosamine residues. CFPS-11 exhibited significant antioxidant activity in a dose-dependent manner and remarkable inhibition activities against α-amylase and α-glucosidase by in vitro assays. These findings indicated that the CFPS-11 from C. fluminea has the potential for development as a health food ingredient.
- Yan, Jing-Kun,Wang, Yao-Yao,Qiu, Wen-Yi,Wu, Li-Xia,Ding, Zhi-Chao,Cai, Wu-Dan
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- Development of a multi-enzymatic desymmetrization and its application for the biosynthesis of L-norvaline from DL-norvaline
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Perindopril is an effective antihypertensive drug in strong demand used to treat hypertension. L-norvaline is a vital intermediate of Perindopril production mainly produced by chemical synthesis with low purity. We developed an environmentally friendly method to produce L-norvaline with high purity based on a desymmetrization process. D-Norvaline was oxidized to the corresponding keto acid by D-amino acid oxidase from the substrate DL-norvaline. Asymmetric hydrogenation of the keto acid to L-norvaline was carried out by leucine dehydrogenase with concomitant oxidation of NADH to NAD+. A NADH regeneration system was introduced by overexpressing a formate dehydrogenase. The unwanted H2O2by-product generated during D-norvaline oxidation was removed by adding catalase. A total of 54.09?g/L of L-norvaline was achieved, with an enantiomeric excess over 99% under optimal conditions, with a 96.7% conversion rate. Our desymmetrization method provides an environmental friendly strategy for the production of enantiomerically pure L-norvaline in the pharmaceutical industry.
- Qi, Yunlong,Yang, Taowei,Zhou, Junping,Zheng, Junxian,Xu, Meijuan,Zhang, Xian,Rao, Zhiming,Yang, Shang-Tian
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p. 104 - 109
(2017/03/23)
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- A novel thyroglobulin-binding lectin from the brown alga Hizikia fusiformis and its antioxidant activities
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A lectin (HFL) was isolated from the brown alga, Hizikia fusiformis, through ion exchange on cellulose DE52 and HPLC with a TSK-gel G4000PWXL column. SDS-PAGE showed that HFL had a molecular mass of 16.1 kDa. The HPLC (with a TSK-gel G4000PWXL column) indicated that HFL is a tetramer in its native state. The total carbohydrate content was 41%. Glucose, galactose and fucose were the monosaccharide units of HFL, and the normalized mol% values were 6, 14 and 80, respectively. HFL contains a large amount of the acidic amino acid, Asx. The β-elimination reaction suggested that the oligosaccharide and peptide moieties of HFL may belong to the N-glucosidic linkage. The amino acid sequences, of about five segments of HFL, were acquired by MALDI-TOF/TOF, and the sequences have no homology with other lectins. HFL was found to agglutinate sheep erythrocytes. The hemagglutination activity was inhibited by thyroglobulin, from bovine thyroid, but not by any of the monosaccharides tested. The lectin reaction was independent of the presence of the divalent cation Ca2+. HFL showed free radical scavenging activity against hydroxyl, DPPH and ABTS+ radicals.
- Wu, Mingjiang,Tong, Changqing,Wu, Yue,Liu, Shuai,Li, Wei
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- Top-down targeted metabolomics reveals a sulfur-containing metabolite with inhibitory activity against angiotensin-converting enzyme in asparagus officinalis
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The discovery of bioactive natural compounds containing sulfur, which is crucial for inhibitory activity against angiotensin-converting enzyme (ACE), is a challenging task in metabolomics. Herein, a new S-containing metabolite, asparaptine (1), was discovered in the spears of Asparagus officinalis by targeted metabolomics using mass spectrometry for S-containing metabolites. The contribution ratio (2.2%) to the IC50 value in the crude extract showed that asparaptine (1) is a new ACE inhibitor.
- Nakabayashi, Ryo,Yang, Zhigang,Nishizawa, Tomoko,Mori, Tetsuya,Saito, Kazuki
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p. 1179 - 1183
(2015/06/02)
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- Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate
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Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys10 reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides.
- Hara, Toshiaki,Purwati, Euis Maras,Tainosyo, Akira,Kawakami, Toru,Hojo, Hironobu,Aimoto, Saburo
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p. 765 - 769
(2015/09/21)
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- Enantiospecific C-H Activation Using Ruthenium Nanocatalysts
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The activation of C-H bonds has revolutionized modern synthetic chemistry. However, no general strategy for enantiospecific C-H activation has been developed to date. We herein report an enantiospecific C-H activation reaction followed by deuterium incorporation at stereogenic centers. Mechanistic studies suggest that the selectivity for the α-position of the directing heteroatom results from a four-membered dimetallacycle as the key intermediate. This work paves the way to novel molecular chemistry on nanoparticles.
- Taglang, Céline,Martínez-Prieto, Luis Miguel,Del Rosal, Iker,Maron, Laurent,Poteau, Romuald,Philippot, Karine,Chaudret, Bruno,Perato, Serge,Sam Lone, Ana?s,Puente, Céline,Dugave, Christophe,Rousseau, Bernard,Pieters, Grégory
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supporting information
p. 10474 - 10477
(2015/09/02)
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- Characterization of two putative prolinases (PepR1 and PepR2) from Lactobacillus plantarum WCFS1: Occurrence of two isozymes with structural similarity and different catalytic properties
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Two putative prolinases (PepR1 and PepR2) of Lactobacillus plantarum WCSF1 share 48.5% amino acid sequence identity (55.5% at the DNA level); however, PepR1 exhibits over 80% identity at the protein level with other lactobacilli prolinases while PepR2 exhibits only 51% or less identity. In this study, the putative genes were overexpressed in Escherichia coli, purified to gel electrophoretic homogeneity, and then characterized. Purified PepR1 and PepR2 hydrolysed Pro-Xaa dipeptide substrates at similar rates, proving their nature as prolinases. Structural analyses using circular dichroism, dynamic light scattering, gel filtration, and molecular modelling revealed that the two prolinases have similar structural characteristics: high β-sheet content, homotetrameric structure, and similar folding to the PepI/PepL/PepR peptidase family. However, kinetic and thermodynamic analyses of PepR1 and PepR2 indicated differences in many aspects: optimum temperatures (25 and 30 °C, respectively), optimum pH (pH 7.5 and 8.0, respectively), substrate specificities (high stringency of PepR2), kinetic parameters, and thermal stability (29 and 48 °C, respectively). Also, these prolinases behaved differently towards inhibitor treatments, suggesting structural and/or functional differences in their active sites. Differences in the two prolinases would contribute to a diversity of catalytic activities, so that they work together cooperatively and complementarily to hydrolyse proline-containing peptides with broader specificity, working pH, working temperature, and higher efficiency, thus allowing adaptation to a wider range of environments.
- Huang, Yanyu,Tanaka, Takuji
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- METHODS FOR IMPROVING HEALTH IN CANINES
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A method of improving health in a canine includes administering to the canine a nutritional supplement comprising an amino acid secretagogue composition, which stimulates the pituitary gland of the canines to produce growth hormone. The nutritional supplement may be administered orally. The nutritional supplement may comprise L-arginine hydrochloride, Oxo-proline, L-lysine hydrochloride, and cysteine. When desired, the nutritional supplement may consist essentially of L-arginine hydrochloride, Oxo-proline, L-lysine hydrochloride, N-acetyl-L-cysteine, L-glutamine, and schizonepeta powder.
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Page/Page column 3
(2014/04/03)
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- SEPARATING AGENT AND MANUFACTURING METHOD THEREOF
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An embodiment of the present invention is a separating agent wherein a group represented by a chemical formula of: or a group represented by a chemical formula of: is introduced on a surface thereof.
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Paragraph 0067; 0068; 0069; 0070; 0071; 0072;
(2015/01/07)
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- A mild removal of Fmoc group using sodium azide
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A mild method for effectively removing the fluorenylmethoxycarbonyl (Fmoc) group using sodium azide was developed. Without base, sodium azide completely deprotected Nα-Fmoc-amino acids in hours. The solvent-dependent conditions were carefully studied and then optimized by screening different sodium azide amounts and reaction temperatures. A variety of Fmoc-protected amino acids containing residues masked with different protecting groups were efficiently and selectively deprotected by the optimized reaction. Finally, a biologically significant hexapeptide, angiotensin IV, was successfully synthesized by solid phase peptide synthesis using the developed sodium azide method for all Fmoc removals. The base-free condition provides a complement method for Fmoc deprotection in peptide chemistry and modern organic synthesis. Graphical Abstract: [Figure not available: see fulltext.]
- Chen, Chun-Chi,Rajagopal, Basker,Liu, Xuan Yu,Chen, Kuan Lin,Tyan, Yu-Chang,Lin, Fui,Lin, Po-Chiao
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p. 367 - 374
(2014/03/21)
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- Efficient one-step preparation of γ-aminobutyric acid from glucose without an exogenous cofactor by the designed Corynebacterium glutamicum
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Lactobacillus plantarum CCTCC M209102 efficiently produces γ-aminobutyric acid (GABA) from l-glutamate, in which glutamate decarboxylase and pyridoxal kinase are involved in the transformation. Pyridoxal kinase catalyzes ATP-dependent phosphorylation of pyridoxal to produce pyridoxal-5′-phosphate, which is the cofactor required for glutamate decarboxylase to biotransform GABA from l-glutamate. Corynebacterium glutamicum G01 is a good producer of l-glutamate from glucose. However, it cannot yield GABA from l-glutamate due to the absence of glutamate decarboxylase and pyridoxal kinase. In this work, to realize the efficient one-step preparation of GABA from glucose without exogenous pyridoxal-5′-phosphate, the metabolic module from L-glutamate to GABA based on glutamate decarboxylase and pyridoxal kinase in L. plantarum was grafted into C. glutamicum. To further improve the GABA production, the pathways to by-product pools of L-arginine, L-proline and L-lysine were blocked using the insertional mutation technique. The engineered C. glutamicum APLGGP carrying argB::tacgad, proB::tacgad and dapA::tacplk could efficiently convert glucose into GABA in one-step without an exogenous co-factor. In fed-batch cultures, the recombinant C. glutamicum APLGGP produced 70.6 g L-1 GABA at 30 °C and 70 h through a two-stage pH control strategy. To our knowledge, this is the highest reported GABA production using glucose as a substrate, and this designed C. glutamicum should be an excellent candidate for producing GABA on an industrial scale. This work is expected to pave the way to redesign the bioreactor for efficient one-step biosynthesis of GABA from glucose without an exogenous co-factor. the Partner Organisations 2014.
- Zhang, Rongzhen,Yang, Taowei,Rao, Zhiming,Sun, Hongmei,Xu, Meijuan,Zhang, Xian,Xu, Zhenghong,Yang, Shangtian
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p. 4190 - 4197
(2014/11/08)
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- Nostosins, trypsin inhibitors isolated from the terrestrial cyanobacterium Nostoc sp. strain FSN
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Two new trypsin inhibitors, nostosin A (1) and B (2), were isolated from a hydrophilic extract of Nostoc sp. strain FSN, which was collected from a paddy field in the Golestan Province of Iran. Nostosins A (1) and B (2) are composed of three subunits, 2-hydroxy-4-(4-hydroxyphenyl)butanoic acid (Hhpba), l-Ile, and l-argininal (1) or argininol (2). Nostosins A (1) and B (2) exhibited IC50 values of 0.35 and 55 μM against porcine trypsin, respectively, suggesting that the argininal aldehyde group plays a crucial role in the efficient inhibition of trypsin. Molecular docking of nostosin A (1) (449 Da), leupeptin (426 Da, IC50 0.5 μM), and spumigin E (610 Da, IC50 A (1) and leupeptin but only partial binding similarity with spumigin E. The number of hydrogen bonds between ligands and trypsin increased according to the length and size of the ligand molecule, and the docking affinity values followed the measured IC50 values. Nostosin A (1) is the first highly potent three-subunit trypsin inhibitor with potency comparable to the known commercial trypsin inhibitor leupeptin. These findings expand the known diversity of short-chain linear peptide protease inhibitors produced by cyanobacteria.
- Liu, Liwei,Jokela, Jouni,Wahlsten, Matti,Nowruzi, Bahareh,Permi, Perttu,Zhang, Yue Zhou,Xhaard, Henri,Fewer, David P.,Sivonen, Kaarina
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p. 1784 - 1790
(2014/10/16)
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- Three bioactive cyclic dipeptides from the Bacillus sp. N strain associated with entomopathogenic nematode
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In continuation of our search for new bioactive secondary metabolites from Bacillus cereus associated with entomopathogenic nematode (EPN), three cyclic dipeptides (CDPs), cyclo(l-Leu-d-Arg) (1), cyclo(2-hydroxy-Pro-l-Leu) (2), and cyclo(l-Val-l-Pro) (3) were purified from the ethyl acetate extract of B. cereus. The chemical structure of the compounds was identified by 1D, 2D NMR and HR-ESI-MS. Cyclo(l-Leu-d-Arg) recorded best antifungal activity and the highest activity was recorded against Cryptococcus neoformans (1 μg/mL), which is better than the standard antifungal agent amphotericin B. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for finding cell proliferation inhibition and cyclo(l-Leu-d-Arg) recorded significant activity against breast cancer cell line (MDAM-B231) (IC50 value: 25 μM) and the three cyclic dipeptides recorded no toxicity against normal human cell (fore skin (FS) normal fibroblast) up to 50 μM except cyclo(l-Val-l-Pro). Cyclo(l-Leu-d-Arg) induced significant morphological changes and DNA fragmentation associated with apoptosis in MDAM-B231 cells by acridine orange/ethidium bromide staining and flow cytometry analysis. Out of three cyclic dipeptides tested only cyclo(2-hydroxy-Pro-l-Leu) recorded significant antioxidant activity. The hydroxyl radical scavenging activity of cyclo(2-hydroxy-Pro-l-Leu) is greater than BHA, the standard antioxidant agent. Cyclo(l-Leu-d-Arg) was isolated for the first time from a natural source with a d-arginine residue. To the best of our knowledge, this is the first time that the bioactivity of the isolated cyclic dipeptides is reported against medically important fungi and cancer cells. This study is a significant contribution to the knowledge of cyclo(l-Leu-d-Arg) from B. cereus as potential sources of new drugs in the pharmacological industry, especially as potent antifungal and anticancer agent.
- Nishanth, Sasidharan Kumar,Nambisan, Bala,Dileep
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- Probing of primed and unprimed sites of calpains: Design, synthesis and evaluation of epoxysuccinyl-peptide derivatives as selective inhibitors
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Calpains are intracellular cysteine proteases with important physiological functions. Up- or downregulation of their expression can be responsible for several diseases, therefore specific calpain inhibitors may be considered as promising candidates for drug discovery. In this paper we describe the synthesis and characterization of a new class of inhibitors derived from the analysis of amino acid preferences in primed and unprimed sites of calpains by incorporation of l- or d-epoxysuccinyl group (Eps). Amino acids for replacement were chosen by considering the substrate preference of calpain 1 and 2 enzymes. The compounds were characterized by RP-HPLC, amino acid analysis and ESI-MS. Selectivity of the compounds was studied by using calpain 1 and 2; and cathepsin B. We have identified five calpain specific inhibitors with different extent of selectivity. Two of these also exhibited isoform selectivity. Compound NH 2-Thr-Pro-Leu-(d-Eps)-Thr-Pro-Pro-Pro-Ser-NH2 proved to be a calpain 2 enzyme inhibitor with at least 11.8-fold selectivity, while compound NH2-Thr-Pro-Leu-(l-Eps)-Ser-Pro-Pro-Pro-Ser-NH2 possesses calpain 1 enzyme inhibition with at least 4-fold selectivity. The results of molecular modeling calculations suggest that the orientation of the bound inhibitor in the substrate binding cleft is markedly dependent on the stereochemistry of the epoxysuccinyl group.
- Dókus, Levente E.,Menyhárd, Dóra K.,Tantos, ágnes,Hudecz, Ferenc,Bánóczi, Zoltán
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supporting information
p. 274 - 280
(2014/06/24)
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- Biochemical characterisation and assessment of fibril-forming ability of collagens extracted from Bester sturgeon Huso huso × Acipenser ruthenus
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Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS-PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3 °C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen.
- Zhang, Xi,Ookawa, Mika,Tan, Yongkai,Ura, Kazuhiro,Adachi, Shinji,Takagi, Yasuaki
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p. 305 - 312
(2014/05/06)
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- A fluorescence turn on trypsin assay based on aqueous polyfluorene
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A new method based on the electrostatic interaction of a novel anionic water soluble polymer P1 with a positively charged polypeptide Arg6 was developed for a continuous and real time turn on assay for the enzymatic activity of trypsin under alkaline conditions with a limit of detection of 0.17 nM. This method was also able to screen the inhibitors of trypsin. P1 fluorescence intensity was significantly decreased by the positively charged Arg6 due to the electrostatic interaction, whereas the enzymatic action recovered P1 fluorescence due to the fragmentation of Arg6 into small positively charged fragments and these were unable to quench the P1 fluorescence. Therefore, by triggering the fluorescence intensity change, it was possible to assay the enzymatic activity. Use of water soluble conjugated polymer P1 and no labeling on the substrate enhances the utility of this method significantly. The Royal Society of Chemistry.
- Dwivedi, Atul Kumar,Iyer, Parameswar Krishnan
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p. 4005 - 4010
(2013/09/23)
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- SEPARATING AGENT FOR CHROMATOGRAPHY
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A separating agent for chromatography is provided that is useful for the separation of specific compounds, e.g., for the optical resolution of amino acids. This separating agent for chromatography provides a higher productivity and contains a crown ether-like cyclic structure and optically active binaphthyl. This separating agent for chromatography containing a crown ether-like cyclic structure and optically active binaphthyl is provided by introducing a substitution group for binding to carrier into a specific commercially available 1,1′-binaphthyl derivative that has substituents at the 2, 2′, 3, and 3′ positions, then introducing a crown ether-like cyclic structure, and subsequently chemically bonding the binaphthyl derivative to the carrier through the substitution group for binding to carrier.
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Paragraph 0074; 0075
(2013/08/15)
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- Using the 9-BBN group as a transient protective group for the functionalization of reactive chains of α-amino acids
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Achieving chemoselectivity is a longstanding challenge in chemical synthesis. This problem has been addressed using different approaches, but a definitive solution is still pending. For instance, in peptide chemistry, particularly with amino acids containing side chains functionalities with reactivity patterns similar to the main functional groups, such as aspartic and glutamic acids, and lysine and ornithine, specific semi-permanent protecting groups have been employed. The use of 9-borabicyclo[3.3.1]nonane (9-BBN-H) as a transient protective group for the selective protection of α-amino acids, which allows the chemoselective manipulation of the functional groups embedded in the side chains of the molecule, is described.
- Sanchez, Adrian,Calderon, Ernesto,Vazquez, Alfredo
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p. 1364 - 1372
(2013/07/05)
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- Identification of new peptide amides as selective cathepsin L inhibitors: The first step towards selective irreversible inhibitors?
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A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the resulting covalent inhibitors.
- Torkar, Ana,Lenar?i?, Brigita,Lah, Tamara,Dive, Vincent,Devel, Laurent
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supporting information
p. 2968 - 2973
(2013/06/27)
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- Peptide bond hydrolysis catalyzed by the Wells-Dawson Zr(α 2-P2W17O61)2 polyoxometalate
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In this paper we report the first example of peptide hydrolysis catalyzed by a polyoxometalate complex. A series of metal-substituted Wells-Dawson polyoxometalates were synthesized, and their hydrolytic activity toward the peptide bond in glycylglycine (GG) was examined. Among these, the Zr(IV)- and Hf(IV)-substituted ones were the most reactive. Detailed kinetic studies were performed with the Zr(IV)-substituted Wells-Dawson type polyoxometalate K 15H[Zr(α2-P2W17O 61)2]·25H2O which was shown to act as a catalyst for the hydrolysis of the peptide bond in GG. The speciation of K 15H[Zr(α2-P2W17O 61)2]·25H2O which is highly dependent on the pD, concentration, and temperature of the solution, was fully determined with the help of 31P NMR spectroscopy and its influence on the GG hydrolysis rate was examined. The highest reaction rate (kobs = 9.2 (±0.2) × 10-5 min-1) was observed at pD 5.0 and 60 °C. A 10-fold excess of GG was hydrolyzed in the presence of K 15H[Zr(α2-P2W17O 61)2]·25H2O proving the principles of catalysis. 13C NMR data suggested the coordination of GG to the Zr(IV) center in K15H[Zr(α2-P2W 17O61)2]·25H2O via its N-terminal amine group and amide carbonyl oxygen. These findings were confirmed by the inactivity of K15H[Zr(α2-P2W 17O61)2]·25H2O toward the N-blocked analogue acetamidoglycylglycinate and the inhibitory effect of oxalic, malic, and citric acid. Triglycine, tetraglycine, and pentaglycine were also fully hydrolyzed in the presence of K15H[Zr(α2- P2W17O61)2]·25H2O yielding glycine as the final product of hydrolysis. K15H[Zr(α 2-P2W17O61)2] ·25H2O also exhibited hydrolytic activity toward a series of other dipeptides.
- Absillis, Gregory,Parac-Vogt, Tatjana N.
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p. 9902 - 9910,9
(2012/12/11)
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- Microbial enantioselective removal of the N-benzyloxycarbonyl amino protecting group
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In order to deprotect N-carbobenzoxy-l-aminoacids (Cbz-AA) and related compounds, a series of microorganisms was selected from soil by enrichment cultures with Cbz-l-Glu as sole nitrogen source. A lyophilized whole-cell preparation of two Arthrobacter sp. strains grown on Cbz-Glu or Cbz-Gly exhibited a high cleavage activity. The conditions of hydrolysis have been optimized and a quantitative enantioselective deprotection of several Cbz-dl-amino acids was obtained, as well as the deprotection of N-carbamoylester derivatives of several synthetic amino compounds. The preparation of Cbz-d-allylglycine and l-allylglycine in high yield and high optical purity is described as an application of this method.
- Maurs, Michele,Acher, Francine,Azerad, Robert
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- New conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retro-tuftsin derivatives significantly influence their biological activity
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The synthesis and biological activity of new conjugates of muramyl dipeptide (MDP) and nor-muramyl dipeptide (nor-MDP) with tuftsin and retro-tuftsin derivatives containing isopeptide bond between e-amino group of lysine and carboxyl group of simple amino
- Dzierzbicka, Krystyna,Wardowska, Anna,Rogalska, Ma?gorzata,Trzonkowski, Piotr
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body text
p. 217 - 223
(2012/08/29)
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- Streptobactin, a tricatechol-type siderophore from marine-derived streptomyces sp. YM5-799
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A new catechol-type siderophore, streptobactin (1), was isolated from a culture broth of the marine-derived actinomycete Streptomyces sp. YM5-799. The structure of streptobactin was determined by NMR and MS analyses and ESIMS/MS experiments to be a cyclic trimer of benarthin. A dibenarthin (2), a tribenarthin (3), and benarthin (4) were also obtained. The production of 1 was regulated by an iron concentration in the culture. The iron-chelating activity of the compounds was evaluated by the chrome azurol sulfonate assay.
- Matsuo, Yoshihide,Kanoh, Kaneo,Jang, Jae-Hyuk,Adachi, Kyoko,Matsuda, Satoru,Miki, Osamu,Kato, Toshiaki,Shizuri, Yoshikazu
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experimental part
p. 2371 - 2376
(2012/02/01)
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- Modulation of the pharmacological activities of secretory phospholipase A2 from Crotalus durissus cascavella induced by naringin
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In this work we have characterized the action of the naringin, a flavonoid found in grapefruit and known for its various pharmacological effects, which include antioxidant, blood lipid lowering and anticancer activity, on the structure and biochemical activities of a secretory phospholipase A (sPLA2) from Crotalus durissus cascavella, an important protein involved in the releasinge of arachidonic acid in phospholipid membranes. sPLA2 was incubated with naringin (mol:mol) at 37 °C and a discrete reduction in the UV scanning signal and a modification of the circular dichroism spectra were observed after treatment with naringin, suggesting modifications of the secondary structure of the protein. This flavonoid was able to decrease enzymatic activity and some pharmacological effects, such as myonecrosis, platelet aggregation, and neurotoxic activity caused by sPLA2, however, the inflammatory effect was not affected by naringin. In addition, small angle X-ray scattering (SAXS) data were collected for sPLA2 and naringin-treated sPLA2 to evaluate possible modifications of the protein structure. These structural investigations have shown that sPLA2 is an elongated dimer in solution and after treatment with naringin a conformational change in the dimeric configuration was observed. Our results suggest that structural modification may be correlated with the loss of enzymatic activity and alterations in pharmacological properties.
- Santos, Marcelo L.,Toyama, Daniela O.,Oliveira, Simone C. B.,Cotrim, Camila A.,Diz-Filho, Eduardo B. S.,Fagundes, Fabio H. R.,Soares, Veronica C. G.,Aparicio, Ricardo,Toyama, Marcos H.
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experimental part
p. 738 - 761
(2011/04/15)
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- Cytotoxic constituents from the skin of the toad Bufo bufo gargarizans
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To study the chemical composition of the skin of Bufo bufo gargarizans, various chromatographic methods were used in the isolation procedures and the structures of isolated compounds were determined based on NMR and MS analysis. As a result, two new compounds were isolated from its ethanolic extract and characterized as N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-methylurea (1) and 19-oxocinobufotalin 3-adipoylarginine ester (2), together with 11 known compounds. Isolated bufadienolides showed significant inhibition effect against human hepatocellular carcinoma cell line SMMC-7721 in vitro.
- Wu, Fu-Kai,Qiu, Ying-Kun,Zhao, Hu-Yi,Wu, Zhen,Li, Fa-Mei,Jiang, Yong-Tao,Chen, Ji-Yong
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experimental part
p. 111 - 116
(2011/04/15)
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- Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties
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Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn2+-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX18E (Zn2+-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G 298XM300E301N302 motif and one mutant of the HEIS328HX18E motif were expressed in Escherichia coli. All mutations except G298P, G298S, and S328A abolished the aminopeptidase activity. The S328A mutant mimics the sequence of bovine Ap-B Zn2+-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G298S and G298P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl- anions. Moreover, the G298P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G298 plays in the catalytic mechanism of Ap-B. Our results show that G298 is essential to Ap-B activity and participates to the substrate specificity of the enzyme.
- Pham, Viet-La?,Gouzy-Darmon, Cécile,Pernier, Julien,Hanquez, Chantal,Hook, Vivian,Beinfeld, Margery C.,Nicolas, Pierre,Etchebest, Catherine,Foulon, Thierry,Cadel, Sandrine
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experimental part
p. 730 - 741
(2012/05/04)
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- Involvement of glutamine-238 in the substrate specificity of human laeverin/aminopeptidase Q
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Human laeverin/aminopeptidase Q (APQ) is a novel member of the M1 family of zinc aminopeptidases and is specifically expressed on the cell surface of extravillous trophoblasts. In this study, we examined the significance of Gln-238 of laeverin/APQ, a putative S1 site residue, by site-directed mutagenesis for its enzymatic activity and substrate specificity. Replacement of Gln-238 with Ala caused a significant change in substrate specificity rather than a decrease in enzymatic activity. These results indicate that Gln-238 is important for the substrate specificity of laeverin/APQ. In addition, our data suggest that direct electrostatic interaction between substrate and S1 site of the enzyme is not involved in the mutant enzyme's preference for basic amino acids.
- Goto, Yoshikuni,Yoshioka, Rina,Arisaka, Naomi,Hattori, Akira,Tsujimoto, Masafumi
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experimental part
p. 24 - 27
(2012/01/06)
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- Thermodynamical characteristics of the reaction of pyridoxal-5'-phosphate with L-amino acids in aqueous buffer solution
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The reaction of pyridoxal-5'-phosphate with L-isomers of alanine, lysine, arginine, aspartic acid, glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calorimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions. The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine. The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the endothermic effect of the reaction increases. Pleiades Publishing, Ltd., 2011.
- Barannikov,Badelin,Venediktov,Mezhevoi,Guseinov
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scheme or table
p. 16 - 20
(2011/06/18)
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- Structure, mechanism, and substrate profile for Sco3058: The closest bacterial homologue to human renal dipeptidase
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Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of β-lactams, is similar in sequence to a cluster of ～400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of L-Xaa-L-Xaa, L-Xaa-D-Xaa, and D-Xaa-L-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an L-amino acid at the N-terminus and a D-amino acid at the C-terminus. The best substrate identified was L-Arg-D-Asp (kcat/Km = 7.6 x 105 M -1 s-1). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of L-Ala-D-Asp. The enzyme folds as a (β/α)8 barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D2O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for kcat and kcat/Km indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of L/D-Ala-L/D-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.
- Cummings, Jennifer A.,Nguyen, Tinh T.,Fedorov, Alexander A.,Kolb, Peter,Xu, Chengfu,Fedorov, Elena V.,Shoichet, Brian K.,Barondeau, David P.,Almo, Steven C.,Raushel, Frank M.
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experimental part
p. 611 - 622
(2011/01/04)
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- Studies on cytotoxic constituents from the skin of the toad Bufo bufo gargarizans
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To study the chemical composition of the skin of Bufo bufo gargarizans, many kinds of chromatography methods were used in the isolation procedures, while the structures of isolated compounds were determined on the basis of their NMR and MS spectral analysis. As a result, two new compounds were isolated from its ethanolic extract and characterized as cinobufotalin 3-nonanedioylarginine ester (8) and bufotalin 3-pimeloylarginine ester (14). Furthermore, 13 known compounds were obtained. Isolated bufadienolides showed significant inhibition effect against SMMC-7721 cell lines in vitro.
- Zhao, Hu-Yi,Wu, Fu-Kai,Qiu, Ying-Kun,Wu, Zhen,Jiang, Yong-Tao,Chen, Ji-Yong
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experimental part
p. 793 - 800
(2010/12/20)
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- Development of a high throughput screening tool for biotransformations utilising a thermophilic l-aminoacylase enzyme
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Micro-reactors containing a monolith-immobilised thermophilic l-aminoacylase, from Thermococcus litoralis, have been developed for use in biotransformation reactions and a study has been carried out to investigate the stereospecificity and stability of the immobilised enzyme. The potential to use the developed micro-reactors as a tool for rapid screening of enzyme specificity was demonstrated, confirming that the l-aminoacylase showed a similar substrate specificity to that previously reported of the free enzyme. From this baseline, the technique was employed as a tool to evaluate potential unreported substrates with N-benzoyl- (l-threonine, l-leucine and l-arginine) and N-acetyl- (d,l-serine, d,l-leucine, l-tyrosine and l-lysine) protecting groups. The order of preferred substrates was found to be Phe > Thr > Leu > Arg for N-benzoyl substrates and Phe ? Ser > Leu > Met > Tyr > Trp for N-acetyl substrates. It was found that by using the micro-reactor a significantly smaller quantity of enzyme and substrates was required. It was shown that the micro-reactors were still operational in the presence of selected organic solvents, such as ethanol, methanol, acetone, dimethylformamide (DMF) and dimethylsulfoxide (DMSO). The results indicated that a combination of a small amount of an appropriate solvent (5% DMSO) and a higher reaction temperature could be employed in biotransformations where substrate solubility was an issue.
- Ngamsom,Hickey,Greenway,Littlechild,Watts,Wiles
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scheme or table
p. 81 - 86
(2010/10/21)
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- PROCESS FOR STRAIGHTENING KERATIN FIBRES WITH A HEATING MEANS AND DENATURING AGENTS
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The invention relates to a process for straightening keratin fibres, comprising: (i) a step in which a straightening composition containing at least two denaturing agents is applied to the keratin fibres, (ii) a step in which the temperature of the keratin fibres is raised, using a heating means, to a temperature of between 110 and 250° C.
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- COMPOSITIONS AND METHODS FOR CYCLOFRUCTANS AS SEPARATION AGENTS
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The present invention relates to derivatized cyclofructan compounds, compositions comprising derivatized cyclofructan compounds, and methods of using compositions comprising derivatized cyclofructan compounds for chromatographic separations of chemical species, including enantiomers. Said compositions may comprise a solid support and/or polymers comprising derivatized cyclofructan compounds.
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Page/Page column 45-49; 61
(2010/12/31)
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- Characterization of a leucine aminopeptidase from Toxoplasma gondii
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The M17 family leucine aminopeptidase (LAP) hydrolyzes amino acids from the N-terminus of peptides. Many LAPs from parasitic protozoa, including Plasmodium, Trypanosoma, and Leishmania, have been intensely investigated because of their crucial roles in parasite biology. In this study, the functional recombinant Toxoplasma gondii LAP (rTgLAP) was expressed in Escherichia coli, and its enzymatic activity against synthetic substrates for aminopeptidase, as well as cellular localization, was determined. The activity was strongly dependent on metal divalent cations, and was inhibited by bestatin, which is an inhibitor for metalloprotease. Our results indicated that TgLAP is a functional aminopeptidase in the cytoplasm of T. gondii.
- Jia, Honglin,Nishikawa, Yoshifumi,Luo, Yuzi,Yamagishi, Junya,Sugimoto, Chihiro,Xuan, Xuenan
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experimental part
p. 1 - 6
(2011/10/12)
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- Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran
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Food-derived bioactive peptides promote functional activity against diseases and present as nutraceutical agents. The purpose of our research was to isolate and fully characterize peptide(s) derived from rice bran having anti-cancer properties. Gastrointestinal juices resistant peptide fractions were initially generated from heat stabilized de-fatted rice bran from which a cell titer assay that uses a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium, inner salt; (MTS)] and the electron coupling reagent, phenazine methosulfate. Ion-exchange chromatography elutes that showed anti-cancer properties were further purified to liberate pure peptide. The pure peptide at 600-700 μg/mL dose caused 84% inhibition to colon cancer cells (Caco-2, HCT-116) growth, 80% to breast cancer cells (MCF-7, MDA-MB-231) growth and 84% to liver cancer cells (HepG-2) growth. Mass spectrometry analysis and de novo sequencing revealed the sequence of Glu-Gln-Arg-Pro-Arg for the peptide with a molecular mass of 685.378 Da. A novel pentapeptide was isolated from rice bran to possess cancer growth inhibitory properties on colon, breast, lung and liver cancer cells. This peptide could serve as a nutraceutical agent against cancer.
- Kannan, Arvind,Hettiarachchy, Navam S.,Lay, Jackson O.,Liyanage, Rohana
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experimental part
p. 1629 - 1634
(2010/11/16)
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- Proline-containing cyclopeptides from the marine sponge phakellia fusca
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Four new cyclopeptides, phakellistatins 15-18 (2-5), together with five known cyclopeptides, phakellistatin 13 (1), hymenistatin 1, and hymenamides G, H, and J, were isolated from the South China Sea sponge Phakellia fusca. Their structures were elucidate
- Zhang, Hong-Jun,Yi, Yang-Hua,Yang, Gen-Jin,Hu, Min-Ye,Cao, Gui-Dong,Yang, Fan,Lin, Hou-Wen
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experimental part
p. 650 - 655
(2010/09/03)
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- Carboxypeptidase from Streptomyces bikiniensis: Primary structure, isolation, and properties
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A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0-7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).
- Serkina,Zalunin,Levitin,Voejkova,Tyaglov,Novikova,Emeljanova,Konstantinova,Chestukhina
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experimental part
p. 1032 - 1038
(2011/11/06)
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