- Synthesis and Reaction of a New Chiral Pyridoxamine Analogue; Some Doubt about the Stereochemical Process Tentatively Proposed for a Nonenzymatic Transamination Reaction
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A pyridoxamine analogue-like chiral pyridinophane with two sulfonyl groups in the bridging chain, (S)-15-aminomethyl-14-hydroxy-2,8-dithia(2,5)pyridinophane S,S,S',S'-tetraoxide ((S)-7), was prepared by oxidation of the sulfide precursor, (S)-2.The amino group was successfully transferred from (S)-7 to several 2-oxo carboxylic acids in methanol at room temperature in the presence of one-half equimolecular zinc(II) ion, giving (R)-amino acids in excess.The reaction rates of this nonenzymatic transamination using chiral (S)-7 were much smaller than those of the corresponding reaction using chiral (S)-2.The enantiomeric excess of the amino acids obtained through the reactions of (S)-7 was compared with those of (S)-2, showing that (S)-7-was more efficient than (S)-2 for the preparation of (R)-alanine, but was less than that of (S)-2 for the preparations of (R)-valine, (R)-leucine, and (R)-phenylalanine.These results aroused some doubt about the previous explanation of the stereochemical features of such nonenzymatic transamination reactions.
- Ando, Makoto,Kuzuhara, Hiroyoshi
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- Real-time monitoring of D-Ala-D-Ala dipeptidase activity of VanX in living bacteria by isothermal titration calorimetry
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The D,D-dipeptidase enzyme VanX is the main cause of vancomycin resistance in gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX hydrolyzing D-Ala-D-Ala and the inhibition constant of D-cysteine inhibitor yielded KM of 0.10 mM, kcat of 11.5 s?1, and Ki of 18.8 μM, which are consistent with the data from ninhydrin/Cd(II)assays. Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE was inhibited by D-cysteine with IC50 values of 29.8 and 28.6 μM, respectively. Also, the total heat from D-Ala-D-Ala (4 mM)hydrolysis decreases strongly (in absolute value)from 1.26 mJ for VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512 μg/mL for VRE and the much smaller value of 4 μg/mL for E. faecalis. The ITC approach proposed here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify drug resistant bacteria.
- Lv, Miao,Zhang, Yue-Juan,Zhou, Fan,Ge, Ying,Zhao, Mu-Han,Liu, Ya,Yang, Ke-Wu
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- Dioncophylline E from Dioncophyllum thollonii, the first 7,3′-coupled dioncophyllaceous naphthylisoquinoline alkaloid
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The isolation and structural elucidation of dioncophylline E, a novel naphthylisoquinoline alkaloid from the rare West African liana Dioncophyllwn thollonii, is described. The alkaloid displays an unusual 7,3′-linkage between the naphthalene and the isoquinoline portions. Due to the resulting medium degree of steric hindrance exerted by the ortho-substituents next to the biaryl axis, the compound undergoes slow rotation about the axis at room temperature and thus is the first such alkaloid that occurs as a mixture of two configurationally semi-stable atropo-diastereomers. Dioncophylline E exhibits good antimalarial activity against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum while its antitrypanosomal activity against Trypanosoma cruzi is weak and that against T. brucei rhodesiense is moderate. Furthermore, four additional naphthylisoquinoline alkaloids previously known from the related plant species Triphyophyllum peltatum, have been identified in D. thollonii.
- Bringmann, Gerhard,Messer, Kim,Wolf, Kristina,Muehlbacher, Joerg,Gruene, Matthias,Brun, Reto,Louis, Adriaan M.
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- Hydrolysis of acetyl-methionine-containing dipeptides promoted by palladium(II) complexes containing methionine-amino acids as ligands
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The [Pd(N,S-Met-a'a'H)(N,S-AcMet-aaH)] (a'a'H and aaH = amino acids) was characterized by electrospray ionization mass spectrometer(ESI-MS) and 1H NMR, in which Met-a'a'H, as ligand, coordinates to Pd(II) via thioether and terminal amino group, and AcMet-aaH, as substrate, coordinates to Pd(II) via thioether and deprotonated amide nitrogen of methionine. The Met-a'a' bond in ligand is intact, the Met-aa bond in substrate, however, is activated toward hydrolysis The difference in hydrolysis behavior between ligand and substrate may be due to a fused six-membered and five-membered ring formation via thioether, deprotonated amide nitrogen and carbonyl oxygen of methionine residue in substrate.
- Luo, Xuemei,Chen, Xiaohua,Song, Yongcheng,Zhu, Longgen
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- Polyoxypeptin isolated from Streptomyces: A bioactive cyclic depsipeptide containing the novel amino acid 3-hydroxy-3-methylproline
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Polyoxypeptin, a potent inducer of apoptosis in human pancreatic carcinoma cells, was isolated from an ethyl acetate extract of a Streptomyces culture broth. Structural determination by 2D-NMR and X-ray crystallographic analysis revealed that it is a novel cyclic hexadepsipeptide containing five hydroxylated amino acids. The unusual and hitherto unreported amino acid 3-hydroxy-3-methylproline was one of them.
- Umezawa, Kazuo,Nakazawa, Kumi,Uemura, Toshio,Ikeda, Yoko,Kondo, Shinichi,Naganawa, Hiroshi,Kinoshita, Naoko,Hashizume, Hideki,Hamada, Masa,Takeuchi, Tomio,Ohba, Shigeru
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- Sinefungin VA and dehydrosinefungin V, new antitrypanosomal antibiotics produced by Streptomyces sp. K05-0178
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Two new nucleotide antibiotics, named sinefungin VA and dehydrosinefungin V, were separated by cation exchange column chromatography and purified by HPLC from the culture broth of Streptomyces sp. K05-0178, together with the known antibiotics, sinefungin, dehydrosinefungin and KSA-9342. The structures of the two novel sinefungin analogs were elucidated by spectroscopic studies, including various NMR and advanced peptide chemical methods. Sinefungin VA consists of adenosine and ornithylvalylalanine, whereas dehydrosinefungin V consists of 4′,5′-dehydroadenosine and ornithylvaline. Sinefungin VA showed potent antitrypanosomal activity with an IC50 value of 0.0026 g ml 1 in vitro without cytotoxicity against MRC-5 cells. Dehydrosinefungin V showed moderate antitrypanosomal activity (IC50=0.15 μg ml-1).
- Niitsuma, Megumi,Hashida, Junko,Iwatsuki, Masato,Mori, Mihoko,Ishiyama, Aki,Namatame, Miyuki,Nishihara-Tsukashima, Aki,Matsumoto, Atsuko,Takahashi, Yoko,Yamada, Haruki,Otoguro, Kazuhiko,Shiomi, Kazuro,Oemura, Satoshi
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- Sustainable synthesis of amino acids by catalytic fixation of molecular dinitrogen and carbon dioxide
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The industrial process of nitrogen fixation is complex and results in a huge economic and environmental impact. It requires a catalyst and high temperature and pressure to induce the rupture of the strong N-N bond and subsequent hydrogenation. On the other hand, carbon dioxide removal from the atmosphere has become a priority objective due to the high amount of global carbon dioxide emissions (i.e. 36 200 million tons in 2015). In this work, we fix nitrogen from N2 and carbon from CO2 and CH4 to obtain both glycine and alanine (d/l racemic mixture), the two simplest amino acids. The synthesis, catalyzed by polarized hydroxyapatite under UV light irradiation and conducted in an inert reaction chamber, starts from a simple gas mixture containing N2, CO2, CH4 and H2O and uses mild reaction conditions. At atmospheric pressure and 95 °C, the glycine and alanine molar yields with respect to CH4 or CO2 are about 1.9% and 1.6%, respectively, but they grow to 3.4% and 2.4%, when the pressure increases to 6 bar and the temperature is maintained at 95 °C. Besides, the minimum temperature required for the successful production of detectable amounts of amino acids is 75 °C. Accordingly, an artificial photosynthetic process has been developed by using an electrophotocatalyst based on hydroxyapatite thermally and electrically stimulated and coated with zirconyl chloride and a phosphonate. The synthesis of amino acids by direct fixation of nitrogen and carbon from gas mixtures opens new avenues regarding the nitrogen fixation for industrial purposes and the recycling of carbon dioxide.
- Rivas, Manuel,Del Valle, Luís J.,Turon, Pau,Alemán, Carlos,Puiggalí, Jordi
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- Argifin, a new chitinase inhibitor, produced by Gliocladium sp. FTD-0668 II. Isolation, physico-chemical properties, and structure elucidation
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A new chitinase inhibitor, named argifin, was isolated from the cultured broth of a fungal strain Gliocladium sp. FTD-0668. Argifin was purified from the cultured mycelium by the combination of cation exchange, anion exchange, adsorption, and gel filtration Chromatographie methods. The structure of argifin was elucidated as cyclo(N-(β-methylcarbamoyl)-L-arginylN-methyl-L-phenyIalanyl-β-L- aspartyl-β-L-aspartyl-D-alanyl) by NMR experiments and other spectroscopic analyses.
- Arai, Noriko,Shiomi, Kazuro,Iwai, Yuzuru,Oemura, Satoshi
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- Artificial metalloenzymes based on protein cavities: Exploring the effect of altering the metal ligand attachment position by site directed mutagenesis
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In an effort to construct catalysts with enzyme-like properties, we are employing a small, cavity-containing protein as a scaffold for the attachment of catalytic groups. In earlier work we demonstrated that a phenanthroline ligand could be introduced into the cavity of the protein ALBP and used to catalyze ester hydrolysis. To examine the effect of positioning the phenanthroline catalyst at different locations within the protein cavity, three new constucts - Phen60, Phen72 and Phen104 - were prepared. Each new conjugate was characterized by UV/vis spectroscopy, fluorescence spectroscopy, guanidine hydrochloride denaturation, gel filtration chromatography, and CD spectroscopy to confirm the preparation of the desired contruct. Analysis of reactions containing Ala-OiPr showed that Phen60 catalyzed ester hydrolysis with less selectivity than ALBP-Phen while Phen72 promoted this same reaction with higher selectivity. Reactions with Tyr-OMe were catalyzed with higher selectivity by Phen60 and more rapidly by Phen104. These results demonstrate that both the rates and selectivities of hydrolysis reactions catalyzed by these constructs are dependent on the precise site of attachment of the metal ligand within the protein cavity.
- Davies, Ronald R.,Kuang, Hao,Qi, Dongfeng,Mazhary, Aram,Mayaan, Evelyn,Distefano, Mark D.
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- Alanine racemase of alfalfa seedlings (Medicago sativa L.): First evidence for the presence of an amino acid racemase in plants
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We demonstrated several kinds of d-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition of l- or d-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of l- and d-alanine. The Km and Vmax values of enzyme for l-alanine were 29.6 × 10-3 M and 1.02 mol/s/kg, and those for d-alanine are 12.0 × 10-3 M and 0.44 mol/s/kg, respectively. The Keq value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5′-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5′-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far.
- Ono, Kazutoshi,Yanagida, Kazuki,Oikawa, Tadao,Ogawa, Tadashi,Soda, Kenji
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- The biosynthetic gene cluster of pantocin A provides insights into biosynthesis and a tool for screening
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Peptide origins: Feeding experiments carried out with 14C-labeled amino acids have demonstrated that the bicyclic core of pantocin A is derived from glutamate or glutamine residues. Sequencing and transposon mutagenesis of the 3.5 kb stretch of DNA that enables an Escherichia coli strain to produce pantocin A has identified three major open-reaing frames (paaA, paaB, and paaC) responsible for its construction, and the resistance mechanism (see picture red: inactive, green: active, yellow: reduced activity positions).
- Jin, Mi,Wright, Sandra A. I.,Beer V, Steven,Clardy, Jon
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- Asymmetric electrosynthesis of amino acid using an electrode modified with amino acid oxidase and electron mediator
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Asymmetric synthesis of amino acid has been successfully achieved by electrochemical reduction of keto acid using an electrode on which amino acid oxidase and electron mediator are immobilized. The enantiomer excess closing to 100% was obtained.
- Kawabata, Susumu,Iwata, Naoko,Yoneyama, Hitoshi
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- Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. II. Structure determination
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The structure of goadsporin was determined by using spectroscopic techniques. NMR analysis revealed that goadsporin consists of 19 amino acids, two of which are dehydroalanines (Deala), and six of which are cyclized to oxazoles (Oxz) and thiazoles (Thz) by dehydrative cyclization and dehydrogenation from serine, threonine and cysteine. NMR analysis established seven partial structures, and their sequence was determined by CID-MS/MS. Negative mode FAB-MS/MS gave product ions arising from charge-remote fragmentation that allowed determination of the sequence of the amino acid components as AcNH-Ala-MeOxz-Val-Deala-MeOxz- Ile-Leu-Thz-Ser-Gly-Gly-MeOxz-Leu-Deala-Oxz-Ala-Gly-Thz-Val-OH. The chiral amino acids were determined by the advanced Marfey's method to have L-configurations.
- Igarashi, Yasuhiro,Kan, Yukiko,Fujii, Kiyonaga,Fujita, Tsuyoshi,Harada, Ken-Ichi,Naoki, Hideo,Tabata, Hirokazu,Onaka, Hiroyasu,Furumai, Tamotsu
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- A potent polymer/pyridoxamine enzyme mimic
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An enzyme mimic consisting of pyridoxamines covalently linked to polyethyleneimine carrying long-chain alkyl groups converts pyruvic acid to dl-alanine with as much as an 8000-fold acceleration relative to the reaction with simple pyridoxamine at the same pyridoxamine concentration. The acceleration by polymer is a strong function of the length of the alkyl chains that are appended. The polymer furnishes acid and base groups to catalyze the proton transfers that are involved in transamination. Copyright
- Liu, Lei,Breslow, Ronald
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- Gageopeptins A and B, new inhibitors of zoospore motility of the phytopathogen Phytophthora capsici from a marine-derived bacterium Bacillus sp. 109GGC020
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Abstract The motility of zoospores is critical in the disease cycles of the peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites regulating the motility of zoospores of Phytophthora capsici, we discovered two new inhibitors from the ethyl acetate extract of the fermentation broth of a marine-derived strain Bacillus sp. 109GGC020. The structures of these novel metabolites were elucidated as new cyclic lipopeptides and named gageopeptins A (1) and B (2) by spectroscopic analyses including high resolution MS and extensive 1D and 2D NMR. The stereoconfigurations of 1 and 2 were assigned based on the chemical derivatization studies and reviews of the literature data. Although compounds 1 and 2 impaired the motility of zoospores of P. capsici in dose- and time-dependent manners, compound 1 (IC50 = 1 μg/ml) was an approximately 400-fold stronger motility inhibitor than 2 (IC50 = 400 μg/ml). Interestingly, the zoospores halted by compound 1 were subsequently lysed at higher concentrations (IC50 = 50 μg/ml). Compounds 1 and 2 were also tested against some bacteria and fungi by broth dilution assay, and exhibited moderate antibacterial and good antifungal activities.
- Tareq, Fakir Shahidullah,Hasan, Choudhury M.,Lee, Hyi-Seung,Lee, Yeon-Ju,Lee, Jong Seok,Surovy, Musrat Zahan,Islam, Md. Tofazzal,Shin, Hee Jae
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- Study of relaxation rates of stable paramagnetic centers in gamma-irradiated alanine.
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The stable L-alanine radical induced by gamma-irradiation was examined by electron paramagnetic resonance (EPR), transfer saturation EPR and electron nuclear double resonance (ENDOR) in the temperature region of fast motion of the methyl group (180-320 K). From the obtained spectral line broadening and spectral intensity the correlation time for the methyl rotation was estimated. The complex processes determining the relaxation rate were examined in the same temperature interval. It was shown that important contributions to the relaxation rate arise from non-secular and pseudo-secular types of contributions. The non-secular contribution involves intramolecular dynamics while the pseudo-secular contribution originates from intermolecular motions. The obtained values for the dynamical parameters have been compared with those obtained by pulse EPR methods and by proton nuclear magnetic resonance (NMR) on undamaged crystals.
- Rakvin,Maltar-Strmecki
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- Spiro fused diterpene-indole alkaloids from a creek-bottom-derived Aspergillus terreus
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Four metabolites, teraspiridoles A-D (2-5), formed from the merger of a diterpene and modified indole scaffold were obtained from an Aspergillus terreus isolate. The structures and absolute configurations of these natural products were established using NMR, mass spectrometry, Marfey's method, VCD, and ECD data. Teraspiridole B (3) exhibited weak inhibition of planaria regeneration/survival.
- Cai, Shengxin,Du, Lin,Gerea, Alexandra L.,King, Jarrod B.,You, Jianlan,Cichewicz, Robert H.
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- The Effect of Visible Light on the Catalytic Activity of PLP-Dependent Enzymes
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Pyridoxal 5’-phosphate (PLP)-dependent enzymes are a versatile class of biocatalysts and feature a variety of industrial applications. However, PLP is light sensitive and can cause inactivation of enzymes in certain light conditions. As most of the PLP-dependent enzymes are usually not handled in dark conditions, we evaluated the effect of visible light on the activity of PLP-dependent enzymes during production as well as transformation. We tested four amine transaminases, from Chromobacterium violaceum, Bacillus megaterium, Vibrio fluvialis and a variant from Arthrobacter species as well as two lysine decarboxylases, from Selenomonas ruminantium and the LDCc from Escherichia coli. It appeared that five of these six enzymes suffered from a significant decrease in activity by up to 90 % when handled in laboratory light conditions. Surprisingly, only the amine transaminase variant from Arthrobacter species appeared to be unaffected by light exposure and even showed an activation to 150 % relative activity over the course of 6 h regardless of the light conditions.
- Gerlach, Tim,Nugroho, David Limanhadi,Rother, D?rte
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- Chiral Metal–Organic Framework Hollow Nanospheres for High-Efficiency Enantiomer Separation
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Chiral ZIF-8 hollow nanospheres with d-histidine as part of chiral ligands (denoted as H-d-his-ZIF-8) were prepared for separation of (±)-amine acids. Compared to bulk d-his-ZIF-8 without a hollow cavity, the prepared H-d-his-ZIF-8 showed 15 times higher separation capacity and higher ee values of 90.5 % for alanine, 95.2 % for glutamic acid and 92.6 % for lysine, respectively.
- Wang, Xiaoshi,Zhu, Yanan,Liu, Jian,Liu, Chang,Cao, Changyan,Song, Weiguo
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- Copper(II)-Catalyzed Transamination of Hydrophobic Pyridoxamine with Pyruvic Acid in Functionalized Bilayer Vesicles
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The copper(II)-catalyzed transamination of 2-methyl-3-hydroxy-4-aminomethyl-5-(dodecylthiomethyl)pyridine (C12SPM) with sodium pyruvate was investigated in an aqueous medium at pH 6.8, μ 0.10 (KCl), and 30.0+/-0.1 deg C in the presence of molecular aggregates of N,N-ditetradecyl-Nα--L-alaninamide bromide (N(1+)C5Ala2C14), N,N-ditetradecyl-Nα--L-histidinamide bromide (N(1+)C5His2C14), or hexadecyltrimethylammonium bromide (CTAB).The reaction afforded the corresponding pyridoxal analogue (C12SPL) and alanine as the final products upon addition of edta which liberates the copper(II) ion from the coordination sites of the aldimine Schiff-base.The coordination interaction between the copper(II) ion and C12SPM, which takes place prior to the transamination, was clarified by electronic spectroscopy.The reactivity of the 2:1 (ketimine:Cu(II)) complex was found to be much larger than that of the 1: 1 complex in the molecular assemblies of N(1+)C5Ala2C14 and CTAB, and the formation of the former species was more pronounced in the N(1+)C5Ala2C14 vesicle.The bilayer vesicle formed with N(1+)C5His2C14 allowed the formation of the 1:1 complex in preference to that of the 2:1 complex, and the coordination-free imidazolyl group of the amphiphile effectively catalyzed the isomerization as a general base.
- Murakami, Yukito,Kikuchi, Jun-ichi,Nakano, Akio,Akiyoshi, Kazunari,Imori, Toru
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- O-Seco-RA-XXIV, a possible precursor of an antitumor peptide RA-XXIV, from Rubia cordifolia L.
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O-Seco-RA-XXIV, a new cyclic peptide, cyclo-(d-alanyl-l-glutaminyl-N,O- dimethyl-l-tyrosyl-l-alanyl-N-methyl-l-tyrosyl-N-methyl-l-tyrosyl), was isolated from the roots of Rubia cordifolia L. along with RA-XXIV. Its structure and relative stereochemistry were determined by interpretation of the spectroscopic data and X-ray crystallography, and its absolute stereochemistry by the Marfey's amino acid analysis of its acid hydrolysate. Isolation of the two peptides from the same plant source may indicate that O-seco-RA-XXIV is a possible precursor of RA-XXIV and that the formation of the diphenyl ether linkage in the cycloisodityrosine moiety is to be formed after the formation of the cyclohexapeptide chain in this series of peptides.
- Hitotsuyanagi, Yukio,Kusano, Jun-Ichi,Kim, Ik-Hwi,Hasuda, Tomoyo,Fukaya, Haruhiko,Takeya, Koichi
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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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- Mirabamides E-H, HIV-inhibitory depsipeptides from the sponge Stelletta clavosa
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Four new depsipeptides, mirabamides E-H (1-4), and the known depsipeptide mirabamide C (5) have been isolated from the sponge Stelletta clavosa, collected from the Torres Strait. The planar structures were determined on the basis of extensive 1D and 2D NMR and HRESIMS. The absolute configurations were established by the advanced Marfey's method, NMR, and GC-MS. The four new compounds all showed strong inhibition of HIV-1 in a neutralization assay with IC50 values of 121, 62, 68, and 41 nM, respectively.
- Lu, Zhenyu,Van Wagoner, Ryan M.,Harper, Mary Kay,Baker, Heather L.,Hooper, John N. A.,Bewley, Carole A.,Ireland, Chris M.
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- Diketopiperazine-Type Alkaloids from a Deep-Sea-Derived Aspergillus puniceus Fungus and Their Effects on Liver X Receptor α
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Eight new diketopiperazine-type alkaloids including four oxepin-containing diketopiperazine-type alkaloids, oxepinamides H-K (1-4), and four 4-quinazolinone alkaloids, puniceloids A-D (5-8), together with two known analogues (9 and 10), were isolated from the culture broth extracts of the deep-sea-derived fungus Aspergillus puniceus SCSIO z021. Their structures were elucidated by spectroscopic analyses, and their absolute configurations were determined by Marfey's method along with comparison of their specific rotations and ECD spectra. The absolute configurations of 4 and 5 were further confirmed by a single-crystal X-ray diffraction analysis. Compounds 1-8 showed significant transcriptional activation of liver X receptor α with EC50 values of 1.7-50 μM, and 7 and 8 were the most potent agonists.
- Liang, Xiao,Zhang, Xuelian,Lu, Xinhua,Zheng, Zhihui,Ma, Xuan,Qi, Shuhua
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- Rapid, Heterogeneous Biocatalytic Hydrogenation and Deuteration in a Continuous Flow Reactor
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The high selectivity of biocatalysis offers a valuable method for greener, more efficient production of enantiopure molecules. Operating immobilised enzymes in flow reactors can improve the productivity and handling of biocatalysts, and using H2 gas to drive redox enzymes bridges the gap to more traditional metal-catalysed hydrogenation chemistry. Herein, we describe examples of H2-driven heterogeneous biocatalysis in flow employing enzymes immobilised on a carbon nanotube column, achieving near-quantitative conversion in 2 gas as a clean reductant, in a completely atom-efficient process. The flow system is demonstrated for cofactor conversion, reductive amination and ketone reduction, and then extended to biocatalytic deuteration for the selective production of isotopically labelled chemicals.
- Thompson, Lisa A.,Rowbotham, Jack S.,Nicholson, Jake H.,Ramirez, Miguel A.,Zor, Ceren,Reeve, Holly A.,Grobert, Nicole,Vincent, Kylie A.
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- Zelkovamycins B-E, Cyclic Octapeptides Containing Rare Amino Acid Residues from an Endophytic Kitasatospora sp
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Four unusual cyclopeptides, zelkovamycins B-E (1-4), were isolated from an endophytic Kitasatospora sp. Zelkovamycin B was featured by an unprecedented 3-methyl-5-hydroxypyrrolidine-2,4-dione ring system linked to the cyclopeptide skeleton. Their structures and full configurations were established by spectroscopic analysis, Marfey's method, and NMR calculations. A plausible biosynthetic pathway for zelkovamycins was proposed based on gene cluster analysis. Zelkovamycin E displayed potent inhibitory activity against H1N1 influenza A virus.
- Cen, Shan,Connolly, Jack A.,Gan, Maoluo,Goss, Rebecca J. M.,Hao, Xiaomeng,Liu, Yufeng,Wang, Yujia,Yu, Jiaqing,Yu, Liyan,Zhang, Yuqin
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- POLYACRYLIC CROSSLINKED RESINS WITH PENDANT CHIRALITY AS AUXILIARY IN SUPPORTED ASYMMETRIC SYNTHESIS
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A new approach is given in polymer assisted asymmetric synthesis, using a polyacrylic crosslinked polymer with pendant chirality as chiral auxiliary.The method is applied to the asymmetric synthesis of aminoacids.
- Calmes, Monique,Daunis, Jacques,Jacquier, Robert,Nkusi, Gerard,Verducci, Jean,Viallefont, Philippe
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- A one-pot enantioselective chemo-enzymatic synthesis of amino acids in water
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The combination of immobilised Rh-Mono-Phos (1-AlTUD-1) and acylase I afforded a chemoenzymatic, one-pot process for the enantioselective synthesis of amino acids in water, without the need for isolation of intermediates. In addition, the enzymatic hydrolysis increases the enantiopurity of the product from 95% ee to >98% ee. Compatibility studies revealed that for optimum results compartmentalisation of the catalysts is required.
- Simons, Chretien,Hanefeld, Ulf,Arends, Isabel W. C. E.,Maschmeyer, Thomas,Sheldon, Roger A.
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- Chemical constituents isolated from Antarctic marine-derived Aspergillus sp. SF-5976 and their anti-inflammatory effects in LPS-stimulated RAW 264.7 and BV2 cells
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Three new benzaldehydes (1–3) and two new dioxopiperazine alkaloids (4 and 5), along with 23 known constituents, were isolated from Antarctic marine-derived Aspergillus sp. SF-5976. Their structures were determined by spectroscopic and chemical methods. Among them, 20 compounds showed inhibitory effects toward lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages with IC50 values of 7.3–77.4 μM, while 22 compounds did in BV2 microglia with those of 4.6–72.3 μM. Furthermore, the effects of the newly isolated compounds on LPS-stimulated inducible NO synthase (iNOS) expression were investigated, and 1–3, and 5 inhibited iNOS in RAW 264.7 cells, while 1–5 did in BV2 cells. Also, 1–3 and 5 inhibited LPS-induced prostaglandin E2 production with IC50 values of 9.1–66.8 μM in RAW 264.7 macrophages by suppressing cyclooxygenase-2 (COX-2), while in BV2 microglia, 1–5 showed activities with those of 2.8–49.4 μM.
- Kwon, Jaeyoung,Lee, Hyaemin,Ko, Wonmin,Kim, Dong-Cheol,Kim, Kwan-Woo,Kwon, Hak Cheol,Guo, Yuanqiang,Sohn, Jae Hak,Yim, Joung Han,Kim, Youn-Chul,Oh, Hyuncheol,Lee, Dongho
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- H2-Driven biocatalytic hydrogenation in continuous flow using enzyme-modified carbon nanotube columns
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We describe the implementation of a system of immobilised enzymes for H2-driven NADH recycling coupled to a selective biotransformation to enable H2-driven biocatalysis in flow. This approach represents a platform that can be optimised for a wide range of hydrogenation steps and is shown here for enantioselective ketone reduction and reductive amination.
- Zor, Ceren,Reeve, Holly A.,Quinson, Jonathan,Thompson, Lisa A.,Lonsdale, Thomas H.,Dillon, Frank,Grobert, Nicole,Vincent, Kylie A.
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- Heterogeneous Photosynthetic Production of Amino Acids at Pt/TiO2 Suspensions by Near Ultraviolet Light
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Studies of the previously reported production of amino acids (glycine, alanine, serine, aspartic acid, glutamic acid) from methane, ammonia, and water in contact with irradiated suspensions of Pt/TiO2 were extended.Products were analyzed by LC, HPLC, and coupled GC/mass spectroscopy.Analysis of product mixtures also shows the presence of MeOH, EtOH, and CH3NH2 as products of the photoprocess.Experiments with nitrogen-15 labeled ammonia showed that the nitrogen in the amino acids originated with the NH3 rather than from contaminants.Amino acids were also produced from the CH4-NH3-H2O mixture during decomposition of hydrogen peroxide at Pt foil.A mechanism for the reaction based on free-radical reactions initiated by hydroxyl radical produced at the irradiated catalyst is proposed.
- Dunn, Wendell W.,Aikawa, Yosihiro,Bard, Allen J.
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- Ancistrobrevines E-J and related naphthylisoquinoline alkaloids from the West African liana Ancistrocladus abbreviatus with inhibitory activities against Plasmodium falciparum and PANC-1 human pancreatic cancer cells
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From the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae), ten new naphthylisoquinoline alkaloids (7a, 7b, 8a, 8b, and 9–14), displaying three different coupling types (5,1′, 5,8′, and 7,8′), were isolated, among them a series of five 5,1′-linked representatives and four metabolites belonging to the rare group of 7,8′-coupled alkaloids. Two of the alkaloids, the ancistrobrevines I (13) and J (14), are only the fourth and fifth examples of 7,8′-linked naphthyldihydroisoquinolines ever found in nature. The stereostructures of the new plant metabolites were determined by spectroscopic, chemical (oxidative degradation), and chiroptical (electronic circular dichroism) methods. For the assignment of the axial configuration of 13 and 14 relative to the stereocenter at C-3, which is too far away for significant NOE long-range interactions, these 7,8′-coupled naphthyldihydroisoquinolines were stereoselectively converted into the respective cis-configured tetrahydroisoquinoline analogs. The newly generated ‘auxiliary’ stereocenter at C-1 permitted decisive NOE interactions between the isoquinoline and the naphthalene parts, and thus a reliable attribution of the axial configuration of 13 and 14. In addition, five known compounds (3, 5, 16, 17, and 20), previously discovered in related African and Asian Ancistrocladus species, have now for the first time been identified in A. abbreviatus. All of these alkaloids are S-configured at C-3 and bear an oxygen function at C-6, and are, thus, typical Ancistrocladaceae-type compounds. Some of the alkaloids of A. abbreviatus exhibited promising activities against the malaria parasite Plasmodium falciparum and PANC-1 human pancreatic cancer cells.
- Fayez, Shaimaa,Feineis, Doris,Aké Assi, Laurent,Kaiser, Marcel,Brun, Reto,Awale, Suresh,Bringmann, Gerhard
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- Structural and functional highlights of methionine aminopeptidase 2 from Leishmania donovani
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Methionine aminopeptidase 2 (MAP2) is a principal regulator of apoptosis for Leishmania donovani and a potential candidate for the design and synthesis of novel antileishmanials. The LdMAP2 gene was cloned in pET28a(+)-SUMO vector, expressed in E. coli and then purified by chromatographic methods. It was found to be a monomer and required divalent metal ion for its activity against synthetic substrates with Co(II), Mg(II), Mn(II) and Ni(II) being the major activators. Moreover, Ca(II) showed the tightest binding with Km value of 124.7 ± 9.2 μM, while Co(II) proved most efficient for catalysis with kcat value of 128.1 ± 4 min?1. The naturally occurring aminopeptidase B inhibitor bestatin was found to be a potent inhibitor of LdMAP2 with a Ki value of 0.86 μM. Further, structural studies with circular dichroism (CD) showed an increase in the α-helical and β-sheet contents and a decrease in random coils in LdMAP2 upon interactions with both bestatin and fluorogenic substrates. Finally, structural studies pointed out key differences in the structure of LdMAP2 and HsMAP2 and their interactions with inhibitor bestatin, Ala-AMC, Leu-AMC and Met-AMC. The structural differences of two orthologs and different binding modes with bestatin can be crucial for the development of novel and specific inhibitor against leishmaniasis.
- Bhat, Saleem Yousuf,Dey, Arijit,Qureshi, Insaf A.
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- Alanine racemase free energy profiles from global analyses of progress curves
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Free energy profiles for alanine racemase from Bacillus stearothermophilus have been determined at pH 6.9 and 8.9 from global analysis of racemization progress curves. This required a careful statistical design due to the problems in finding the global minimum in mean square for a system with eight adjustable parameters (i.e., the eight rate constants that describe the stepwise chemical mechanism). The free energy profiles obtained through these procedures are supported by independent experimental evidence: (1) steady-state kinetic constants, (2) solvent viscosity dependence, (3) spectral analysis of reaction intermediates, (4) equilibrium overshoots for progress curves measured in D 2O, and (5) the magnitudes of calculated intrinsic kinetic isotope effects. The free energy profiles for the enzyme are compared to those of the uncatalyzed and the PLP catalyzed reactions. At pH 6.9, PLP lowers the free energy of activation for deprotonation by 8.4 kcal/mol, while the inclusion of apoenzyme along with PLP additionally lowers it by 11 kcal/mol.
- Spies, M. Ashley,Woodward, Joshua J.,Watnik, Mitchell R.,Toney, Michael D.
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- Enzymatic characterization and crystal structure of biosynthetic alanine racemase from Pseudomonas aeruginosa PAO1
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Alanine racemase is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that reversibly catalyzes the conversion of L-alanine to D-alanine. D-alanine is an essential constituent in many prokaryotic cell structures. Inhibition of alanine racemase is lethal to prokaryotes, creating an attractive target for designing antibacterial drugs. Here we report the crystal structure of biosynthetic alanine racemase (Alr) from a pathogenic bacteria Pseudomonas aeruginosa PAO1. Structural studies showed that P. aeruginosa Alr (PaAlr) adopts a conserved homodimer structure. A guest substrate D-lysine was observed in the active site and refined to dual-conformation. Two buffer ions, malonate and acetate, were bound in the proximity to D-lysine. Biochemical characterization revealed the optimal reaction conditions for PaAlr.
- Dong, Hui,Han, Qingqing,Guo, Yu,Ju, Jiansong,Wang, Shanshan,Yuan, Chao,Long, Wei,He, Xin,Xu, Shujing,Li, Sheng
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- Molluscicidal metabolites from an assemblage of Palmyra Atoll cyanobacteria
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Molluscicides can play an important role in the control of schistosomiasis because snails of the genus Biomphalaria act as intermediate hosts for the parasite. Schistosomiasis is one of 13 neglected tropical diseases with high morbidity and mortality that collectively affect one billion of the world's poorest population, mainly in developing countries. Thiopalmyrone (1) and palmyrrolinone (2), metabolites isolated from extracts of a Palmyra Atoll environmental assemblage of two cyanobacteria, cf. Oscillatoria and Hormoscilla spp., represent new and potent molluscicidal chemotypes against Biomphalaria glabrata (LC50 = 8.3 and 6.0 μM, respectively). A slight enhancement in molluscicidal effect (LC50 = 5.0 μM) was observed when these two natural products were utilized as an equimolar binary mixture. (Chemical Equation Presented).
- Pereira, Alban R.,Etzbach, Lena,Engene, Niclas,Mueller, Rolf,Gerwick, William H.
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- Squamins C–F, four cyclopeptides from the seeds of Annona globiflora
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Four cyclic octapeptides, squamins C–F, were isolated from the seeds of Annona globiflora Schltdl. These compounds share part of their amino acid sequence, -Pro-Met(O)-Tyr-Gly-Thr-, with previously reported squamins A and B. Their structures were determined using NMR spectroscopic techniques together with quantum mechanical calculations (QM-NMR), ESI-HRMS data and a modified version of Marfey's chromatographic method. All compounds showed cytotoxic activity against DU-145 (human prostate cancer) and HeLa (human cervical carcinoma) cell lines. Clearly, A. globiflora is an important source of bioactive molecules, which could promote the sustainable exploitation of this undervalued specie.
- Sosa-Rueda, Javier,Domínguez-Meléndez, Vanihamin,Ortiz-Celiseo, Araceli,López-Fentanes, Fernando C.,Cuadrado, Cristina,Fernández, José J.,Daranas, Antonio Hernández,Cen-Pacheco, Francisco
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- Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase
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N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N-bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains—including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site—was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.
- Gu, Zhenghua,Guo, Zitao,Shao, Jun,Shen, Chen,Shi, Yi,Tang, Mengwei,Xin, Yu,Zhang, Liang
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- Rational engineering ofAcinetobacter tandoiiglutamate dehydrogenase for asymmetric synthesis ofl-homoalanine through biocatalytic cascades
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l-Homoalanine, a useful building block for the synthesis of several chiral drugs, is generally synthesized through biocascades using natural amino acids as cheap starting reactants. However, the addition of expensive external cofactors and the low efficiency of leucine dehydrogenases towards the intermediate 2-ketobutyric acid are two major challenges in industrial applications. Herein, a dual cofactor-dependent glutamate dehydrogenase fromAcinetobacter tandoii(AtGluDH) was identified to help make full use of the intracellular pool of cofactors when using whole-cell catalysis. Through reconstruction of the hydrophobic network between the enzyme and the terminal methyl group of the substrate 2-ketobutyric acid, the strict substrate specificity ofAtGluDH towards α-ketoglutarate was successfully changed, and the activity obtained by the most effective mutant (K76L/T180C) was 17.2 times higher than that of the wild-type protein. A three-enzyme co-expression system was successfully constructed in order to help release the mass transfer restriction. Using 1 Ml-threonine, which is close to the solubility limit, we obtained a 99.9% yield ofl-homoalanine in only 3.5 h without adding external coenzymes to the cascade, giving 99.9% ee and a 29.2 g L?1h?1space-time yield. Additionally, the activities of the engineeredAtGluDH towards some other hydrophobic amino acids were also improved to 1.1-11.2 fold. Therefore, the engineering design of some dual cofactor-dependent GluDHs could not only eliminate the low catalytic activity of unnatural substrates but also enhance the cofactor utilization efficiency of these enzymes in industrial applications.
- Diao, Shiqing,Jiang, Shuiqin,Liu, Yan,Sun, Yangyang,Wang, Hualei,Wang, Liuzhu,Wei, Dongzhi
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p. 4208 - 4215
(2021/06/30)
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- Biosynthesis ofl-alanine fromcis-butenedioic anhydride catalyzed by a triple-enzyme cascadeviaa genetically modified strain
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In industry,l-alanine is biosynthesized using fermentation methods or catalyzed froml-aspartic acid by aspartate β-decarboxylase (ASD). In this study, a triple-enzyme system was developed to biosynthesizel-alanine fromcis-butenedioic anhydride, which was cost-efficient and could overcome the shortcomings of fermentation. Maleic acid formed bycis-butenedioic anhydride dissolving in water was transformed tol-alanineviafumaric acid andl-asparagic acid catalyzed by maleate isomerase (MaiA), aspartase (AspA) and ASD, respectively. The enzymatic properties of ASD from different origins were investigated and compared, as ASD was the key enzyme of the triple-enzyme cascade. Based on cofactor dependence and cooperation with the other two enzymes, a suitable ASD was chosen. Two of the three enzymes, MaiA and ASD, were recombinant enzymes cloned into a dual-promoter plasmid for overexpression; another enzyme, AspA, was the genomic enzyme of the host cell, in which AspA was enhanced by a T7promoter. Two fumarases in the host cell genome were deleted to improve the utilization of the intermediate fumaric acid. The conversion of whole-cell catalysis achieved 94.9% in 6 h, and the productivity given in our system was 28.2 g (L h)?1, which was higher than the productivity that had been reported. A catalysis-extraction circulation process for the synthesis ofl-alanine was established based on high-density fermentation, and the wastewater generated by this process was less than 34% of that by the fermentation process. Our results not only established a new green manufacturing process forl-alanine production fromcis-butenedioic anhydride but also provided a promising strategy that could consider both catalytic ability and cell growth burden for multi-enzyme cascade catalysis.
- Cui, Ruizhi,Liu, Zhongmei,Yu, Puyi,Zhou, Li,Zhou, Zhemin
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supporting information
p. 7290 - 7298
(2021/09/28)
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- A Photoregulated Racemase Mimic
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The racemase enzymes convert L-amino acids to their D-isomer. The reaction proceeds through a stepwise deprotonation–reprotonation mechanism that is assisted by a pyridoxal phosphate (PLP) coenzyme. This work reports a PLP–photoswitch–imidazole triad where the racemization reaction can be controlled by light by tweaking the distance between the basic residue and the reaction centre.
- Saha, Monochura,Hossain, Munshi Sahid,Bandyopadhyay, Subhajit
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supporting information
p. 5220 - 5224
(2021/01/18)
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- Mechanistic insight into metal ion-catalyzed transamination
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Several classes of biological reactions that are mediated by an enzyme and a co-factor can occur, to a slower extent, not only without the enzyme but even without the co-factor, under catalysis by metal ions. This observation has led to the proposal that metabolic pathways progressively evolved from using inorganic catalysts to using organocatalysts of increasing complexity. Transamination, the biological process by which ammonia is transferred between amino acids and α-keto acids, has a mechanism that has been well studied under enzyme/co-factor catalysis and under co-factor catalysis, but the metal ion-catalyzed variant was generally studied mostly at high temperatures (70-100 °C), and the details of its mechanism remained unclear. Here, we investigate which metal ions catalyze transamination under conditions relevant to biology (pH 7, 20-50 °C) and study the mechanism in detail. Cu2+, Ni2+, Co2+, and V5+ were identified as the most active metal ions under these constraints. Kinetic, stereochemical, and computational studies illuminate the mechanism of the reaction. Cu2+ and Co2+ are found to predominantly speed up the reaction by stabilizing a key imine intermediate. V5+ is found to accelerate the reaction by increasing the acidity of the bound imine. Ni2+ is found to do both to a limited extent. These results show that direct metal ion-catalyzed amino group transfer is highly favored even in the absence of co-factors or protein catalysts under biologically compatible reaction conditions.
- Mayer, Robert J.,Kaur, Harpreet,Rauscher, Sophia A.,Moran, Joseph
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supporting information
p. 19099 - 19111
(2021/11/22)
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- Highly Stable Zr(IV)-Based Metal-Organic Frameworks for Chiral Separation in Reversed-Phase Liquid Chromatography
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Separation of racemic mixtures is of great importance and interest in chemistry and pharmacology. Porous materials including metal-organic frameworks (MOFs) have been widely explored as chiral stationary phases (CSPs) in chiral resolution. However, it remains a challenge to develop new CSPs for reversed-phase high-performance liquid chromatography (RP-HPLC), which is the most popular chromatographic mode and accounts for over 90% of all separations. Here we demonstrated for the first time that highly stable Zr-based MOFs can be efficient CSPs for RP-HPLC. By elaborately designing and synthesizing three tetracarboxylate ligands of enantiopure 1,1′-biphenyl-20-crown-6, we prepared three chiral porous Zr(IV)-MOFs with the framework formula [Zr6O4(OH)8(H2O)4(L)2]. They share the same flu topological structure but channels of different sizes and display excellent tolerance to water, acid, and base. Chiral crown ether moieties are periodically aligned within the framework channels, allowing for stereoselective recognition of guest molecules via supramolecular interactions. Under acidic aqueous eluent conditions, the Zr-MOF-packed HPLC columns provide high resolution, selectivity, and durability for the separation of a variety of model racemates, including unprotected and protected amino acids and N-containing drugs, which are comparable to or even superior to several commercial chiral columns for HPLC separation. DFT calculations suggest that the Zr-MOF provides a confined microenvironment for chiral crown ethers that dictates the separation selectivity.
- Jiang, Hong,Yang, Kuiwei,Zhao, Xiangxiang,Zhang, Wenqiang,Liu, Yan,Jiang, Jianwen,Cui, Yong
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supporting information
p. 390 - 398
(2021/01/13)
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- Inherently chiral dialkyloxy-calix[4]arene acetic acids as enantiodiscriminating additives for high-performance liquid chromatography separation of d,l-amino acids
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Inherently chiral dialkyloxy-calix[4]arene acetic acids with asymmetric placement of substituents on the lower rim of the macrocycle were first studied as enantiodiscriminating additives to the mobile phase MeCN/H2O/HCOOH (75/25/0.02 by volume) in the high-performance liquid chromatography (HPLC) separation of d,l-alanine and d,l-valine on the achiral stationary phase ZORBAX Original CN. The dependence of enantio-binding properties on the position of alkyl groups is demonstrated. The highest resolution (1.65) and enantioselectivity (1.80) were obtained for the 1,2-dipropyloxy-calix[4]arene acetic acid.
- Kalchenko, Olga I.,Trybrat, Oleksandr O.,Yesypenko, Oleksandr A.,Dyakonenko, Viktoriya V.,Shishkina, Svitlana V.,Kalchenko, Vitali I.
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p. 722 - 730
(2021/08/26)
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- 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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- Biocatalysed synthesis of chiral amines: continuous colorimetric assays for mining amine-transaminases
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In the course of our research aimed at the design of new biocatalytic processes for the enantioselective synthesis of chiral amines, we have developed new continuous assays for the screening of amine-transaminase collections. These assays are based on the use of hypotaurine as an irreversible amine donor. This β-aminosulfinic acid is converted upon transamination into 2-oxoethylsulfinic acid, which instantaneously decomposes into acetaldehyde and sulfite ions that can be easily detected by spectrophotometry using Ellman's reagent. Two complementary assays were developed based on this titration method. Firstly, a direct assay allowed detection of various transaminases able to use hypotaurine as an amino donor. In a second coupled assay,l-alanine is used as a generic donor substrate of amine-transaminases and is regenerated using an auxiliary hypotaurine-transaminase. The powerful and complementary nature of both assays was demonstrated through the screening of a collection of 549 amine-transaminases from biodiversity, thus allowing the discovery of a variety of valuable new biocatalysts for use in synthetic processes.
- Gourbeyre, Léa,Heuson, Egon,Charmantray, Franck,Hélaine, Virgil,Debard, Adrien,Petit, Jean-Louis,de Berardinis, Véronique,Gefflaut, Thierry
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p. 904 - 911
(2021/02/26)
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- Synthesis of new amides based on N-Phthaloyl-α-Amino Acids
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N-phthaloyl derivatives of aliphatic α-amino acids were synthesized using phthalanhydride under standard conditions. The optimization reaction carried out by the thermal method to obtain the amides of these N-phthaloyl amino acids resulted in transimitted rather than amidation. The target amides of N-phthaloyl-α-amino acids were obtained by acylation of the amine with the corresponding acid chloroanhydrides in dichloromethane. These results were compared with the results of a similar acylation in a non-polar solvent (benzene). The dependence of the direction of the reaction on the duration of the acylation and the amount of amine used was established. The conditions for the formation of the corresponding N-phthaloyl-α-amino acid amides and asymmetric phthalic acid diamides were found. It is noteworthy that the formation of diamides is directly proportional to the equivalent amount of amine and the duration of the reaction, which makes it possible to purposefully control the synthesis in one reactor.
- Tukhtaev,Yusupov,Vinogradova
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p. 3049 - 3058
(2021/05/28)
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- Targeted Isolation of Asperheptatides from a Coral-Derived Fungus Using LC-MS/MS-Based Molecular Networking and Antitubercular Activities of Modified Cinnamate Derivatives
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Under the guidance of MS/MS-based molecular networking, four new cycloheptapeptides, namely, asperheptatides A-D (1-4), were isolated together with three known analogues, asperversiamide A-C (5-7), from the coral-derived fungus Aspergillus versicolor. The planar structures of the two major compounds, asperheptatides A and B (1 and 2), were determined by comprehensive spectroscopic data analysis. The absolute configurations of the amino acid residues were determined by advanced Marfey's method. The two structurally related trace metabolites, asperheptatides C and D (3 and 4), were characterized by ESI-MS/MS fragmentation methods. A series of new derivatives (8-26) of asperversiamide A (5) were semisynthesized. The antitubercular activities of 1, 2, and 5-26 against Mycobacterium tuberculosis H37Ra were also evaluated. Compounds 9, 13, 23, and 24 showed moderate activities with MIC values of 12.5 μM, representing a potential new class of antitubercular agents.
- Chao, Rong,Hou, Xue-Mei,Xu, Wei-Feng,Hai, Yang,Wei, Mei-Yan,Wang, Chang-Yun,Gu, Yu-Cheng,Shao, Chang-Lun
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- Engineering the large pocket of an (S)-selective transaminase for asymmetric synthesis of (S)-1-amino-1-phenylpropane
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Amine transaminases offer an environmentally benign chiral amine asymmetric synthesis route. However, their catalytic efficiency towards bulky chiral amine asymmetric synthesis is limited by the natural geometric structure of the small pocket, representing a great challenge for industrial applications. Here, we rationally engineered the large binding pocket of an (S)-selective ?-transaminase BPTA fromParaburkholderia phymatumto relieve the inherent restriction caused by the small pocket and efficiently transform the prochiral aryl alkyl ketone 1-propiophenone with a small substituent larger than the methyl group. Based on combined molecular docking and dynamic simulation analyses, we identified a non-classical substrate conformation, located in the active site with steric hindrance and undesired interactions, to be responsible for the low catalytic efficiency. By relieving the steric barrier with W82A, we improved the specific activity by 14-times compared to WT. A p-p stacking interaction was then introduced by M78F and I284F to strengthen the binding affinity with a large binding pocket to balance the undesired interactions generated by F44. T440Q further enhanced the substrate affinity by providing a more hydrophobic and flexible environment close to the active site entry. Finally, we constructed a quadruple variant M78F/W82A/I284F/T440Q to generate the most productive substrate conformation. The 1-propiophenone catalytic efficiency of the mutant was enhanced by more than 470-times in terms ofkcat/KM, and the conversion increased from 1.3 to 94.4% compared with that of WT, without any stereoselectivity loss (ee > 99.9%). Meanwhile, the obtained mutant also showed significant activity improvements towards various aryl alkyl ketones with a small substituent larger than the methyl group ranging between 104- and 230-fold, demonstrating great potential for the efficient synthesis of enantiopure aryl alkyl amines with steric hindrance in the small binding pocket.
- Liu, He,Wang, Hualei,Wei, Dongzhi,Xie, Youyu,Xu, Feng,Xu, Xiangyang,Yang, Lin
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p. 2461 - 2470
(2021/04/22)
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- Leveraging Peptaibol Biosynthetic Promiscuity for Next-Generation Antiplasmodial Therapeutics
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Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC50 25 μM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.
- Lee, Jin Woo,Collins, Jennifer E.,Wendt, Karen L.,Chakrabarti, Debopam,Cichewicz, Robert H.
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supporting information
p. 503 - 517
(2021/03/01)
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- Mechanistic Insight into the Origin of Stereoselectivity in the Ribose-Mediated Strecker Synthesis of Alanine
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Enantioenriched amino acids are produced in a hydrolytic kinetic resolution of racemic aminonitriles mediated by chiral pentose sugars. Experimental kinetic and spectroscopic results combined with DFT computational studies and microkinetic modeling help to identify the nature of the intermediate species and provide insight into the stereoselectivity of their hydrolysis in the prebiotically relevant ribose-alanine system. These studies support a synergistic role for sugars and amino acids in the emergence of homochirality in biological molecules.
- Legnani, Luca,Darù, Andrea,Jones, Alexander X.,Blackmond, Donna G.
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supporting information
p. 7852 - 7858
(2021/05/26)
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- Method for photolysis of amido bonds
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The invention discloses a method for photo-splitting amido bonds, wherein the method is mild in reaction condition and can realize splitting of amido bonds by using illumination. The method for photo-splitting the amido bonds comprises the following steps: reacting 2,4-dinitrofluorobenzene with an amino group of a substance which contains alpha amino acid at the tail end and is shown as a structural formula I to generate a compound 1 represented by a structural formula II; and under light irradiation, carrying out amido bond cleavage reaction on the compound 1, wherein R1 is a side chain group of alpha-amino acid, and R2 is aryl, aliphatic hydrocarbon, -CH(R)-COOH or polypeptide.
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Paragraph 0046; 0048-0049; 0086-0089
(2021/06/26)
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- Stereo-selective synthesis of non-canonical γ-hydroxy-α-amino acids by enzymatic carbon-carbon bond formation
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Carbon-carbon (C-C) bond formation is the fundamental reaction type in organic synthesis. Biocatalytic methods for C-C bond formation have been limited to a few types of enzymes. In this report, we demonstrated the capability of a PLP-dependent enzyme ApUstD performing both C-C bond activation and asymmetric C-C bond formation, which resulted in non-canonical γ-hydroxy-α-amino acids. The reaction showed high efficiency (conversion up to 98%), stereo-selectivity (ratio up to >97:3), a broad substrate scope (25 isolated examples) and significant simplicity. Our results have extended the biocatalytic function of PLP-dependent enzymes for asymmetric C-C formation.
- Zhang, Rui,Tan, Jiamu,Luo, Zhenzhen,Dong, Haihong,Ma, Ningshan,Liao, Cangsong
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p. 7380 - 7385
(2021/11/27)
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- Biosynthesis of a New Fusaoctaxin Virulence Factor in Fusarium graminearum Relies on a Distinct Path to Form a Guanidinoacetyl Starter Unit Priming Nonribosomal Octapeptidyl Assembly
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Fusarium graminearum is a pathogenic fungus causing huge economic losses worldwide via crop infection leading to yield reduction and grain contamination. The process through which the fungal invasion occurs remains poorly understood. We recently characterized fusaoctaxin A in F. graminearum, where this octapeptide virulence factor results from an assembly line encoded in fg3_54, a gene cluster proved to be involved in fungal pathogenicity and host adaptation. Focusing on genes in this cluster that are related to fungal invasiveness but not to the biosynthesis of fusaoctaxin A, we here report the identification and characterization of fusaoctaxin B, a new octapeptide virulence factor with comparable activity in wheat infection. Fusaoctaxin B differs from fusaoctaxin A at the N-terminus by possessing a guanidinoacetic acid (GAA) unit, formation of which depends on the combined activities of the protein products of fgm1-3. Fgm1 is a cytochrome P450 protein that oxygenates l-Arg to 4(R)-hydroxyl-l-Arg in a regio- and stereoselective manner. Then, Cβ-Cγ bond cleavage proceeds in the presence of Fgm3, a pyridoxal-5′-phosphate-dependent lyase, giving guanidinoacetaldehyde and l-Ala. Rather than being directly oxidized to GAA, the guanidine-containing aldehyde undergoes spontaneous cyclization and subsequent enzymatic dehydrogenation to provide glycociamidine, which is linearized by Fgm2, a metallo-dependent amidohydrolase. The GAA path in F. graminearum is distinct from that previously known to involve l-Arg:l-Gly aminidotransferase activity. To provide this nonproteinogenic starter unit that primes nonribosomal octapeptidyl assembly, F. graminearum employs new chemistry to process l-Arg through inert C-H bond activation, selective C-C bond cleavage, cyclization-based alcohol dehydrogenation, and amidohydrolysis-associated linearization.
- Chen, Dandan,Liu, Wen,Tang, Haoyu,Tang, Weihua,Tang, Zhijun,Wang, Wanqiu,Xue, Yufeng
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supporting information
p. 19719 - 19730
(2021/11/30)
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- Structure revision of isocereulide A, an isoform of the food poisoning emetic Bacillus cereus toxin cereulide
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The emetic Bacillus cereus toxin cereulide presents an enormous safety hazard in the food industry, inducing emesis and nausea after the consumption of contaminated foods. Additional to cereulide itself, seven structurally related isoforms, namely the isocereulides A-G, have already been elucidated in their chemical structure and could further be identified in B. cereus contaminated food samples. The newly performed isolation of isocereulide A allowed, for the first time, 1D- and 2D-NMR spectroscopy of a biosynthetically produced isocereulide, revealing results that contradict previous assumptions of an L-O-Leu moiety within its chemical structure. By furthermore applying posthydrolytical dipeptide analysis, amino acid and α-hydroxy acid analysis by means of UPLC-ESITOF- MS, as well as MSn sequencing, the structure of previously reported isocereulide A could be corrected. Instead of the L-O-Leu as assumed to date, one L-O-Ile unit could be verified in the cyclic dodecadepsipeptide, revising the structure of isocereulide A to [(D-O-Leu-D-Ala-L-O-Val-L-Val)2(DO- Leu-D-Ala-L-O-Ile-L-Val)].
- Ehling-Schulz, Monika,Hofmann, Thomas F.,Kranzler, Markus,Stark, Timo D.,Walser, Veronika
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supporting information
(2021/05/31)
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- Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry
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Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also time-consuming. Here, a mass-tagged chiral selector named [d0]/[d5]-estradiol-3-benzoate-17β-chloroformate ([d0]/[d5]-17β-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17β-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (~2 s). A linear calibration curve of enantiomeric excess was also established using [d0]/[d5]-17β-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17β-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of-AAs in complex biological samples. These results indicated that [d0]/[d5]-17β-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.
- Li, Yuling,Zhou, Bowen,Wang, Keke,Zhang, Jing,Sun, Wenjian,Zhang, Li,Guo, Yinlong
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p. 13589 - 13596
(2021/10/21)
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- Structures and Biosynthetic Pathway of Coprisamides C and D, 2-Alkenylcinnamic Acid-Containing Peptides from the Gut Bacterium of the Carrion Beetle Silpha perforata
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Coprisamides C and D (1 and 2) were isolated from a gut bacterium, Micromonospora sp. UTJ3, of the carrion beetle Silpha perforata. Based on the combined analysis of UV, MS, and NMR spectral data, the planar structures of 1 and 2 were elucidated to be unreported derivatives of coprisamides A and B, cyclic depsipeptides bearing a 2-alkenylcinnamic acid unit and the unusual amino acids β-methylaspartic acid and 2,3-diaminopropanoic acid. The absolute configuration of 1 was determined using the advanced Marfey's method, phenylglycine methyl ester derivatization, and J-based configuration analysis. The biosynthetic gene clusters for the coprisamides were investigated based on genomic data from coprisamide-producing strains Micromonospora sp. UTJ3 and Streptomyces sp. SNU533. Coprisamide C (1) was active against the Mycobacterium tuberculosis mc26230 strain.
- Shin, Yern-Hyerk,Ban, Yeon Hee,Kim, Tae Ho,Bae, Eun Seo,Shin, Jongheon,Lee, Sang Kook,Jang, Jichan,Yoon, Yeo Joon,Oh, Dong-Chan
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- Simultaneous Preparation of (S)-2-Aminobutane and d -Alanine or d -Homoalanine via Biocatalytic Transamination at High Substrate Concentration
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(S)-2-Aminobutane, d-alanine, and d-homoalanine are important intermediates for the production of various active pharmaceutical ingredients and food additives. The preparation of these small chiral amine or amino acids with high water solubility still demands searching for efficient methods. In this work, we identified an ω-transaminase (ω-TA) from Sinirhodobacter hungdaonensis (ShdTA) that catalyzed the kinetic resolution of racemic 2-aminobutane at a concentration of 800 mM using pyruvate as the amino acceptor, leading to the simultaneous isolation of enantiopure (S)-2-aminobutane and d-alanine in 46% and 90% yield, respectively. In addition, (S)-2-aminobutane (98% ee) and d-homoalanine (99% ee) were isolated in 45% and 93% yield, respectively, in the kinetic resolution of racemic 2-aminobutane at a concentration of 400 mM coupled with deamination of l-threonine by threonine deaminase. We thus developed a biocatalytic process for the practical synthesis of these valuable small chiral amine and d-amino acids.
- Li, Jianjiong,Wang, Yingang,Wu, Qiaqing,Yao, Peiyuan,Yu, Shanshan,Zhu, Dunming
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supporting information
(2022/03/01)
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- Highly Efficient Synthesis of Amino Acids by Amination of Bio-Derived Hydroxy Acids with Ammonia over Ru Supported on N-Doped Carbon Nanotubes
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The amino acids have extensive applications, and their productions from biomass-derived feedstocks are very attractive. In this work, the synthesis of amino acids by amination of bio-derived hydroxy acids with ammonia over different metallic nano-catalysts supported on various supports is studied. It is found that Ru nano-catalysts on the nitrogen-doped carbon nanotubes (Ru/N?CNTs) have an outstanding performance for the reaction. Different hydroxy acids can be catalytically converted into the corresponding amino acids with yields up to 70.0 % under mild conditions, which is higher than those reported. The reasons for the high efficiency of the catalyst are investigated, and the reaction pathway is proposed on the basis of control experiments.
- Xie, Zhenbing,Chen, Bingfeng,Peng, Fangfang,Liu, Mingyang,Liu, Huizhen,Yang, Guanying,Han, Buxing
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p. 5683 - 5689
(2020/09/21)
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- Semi-rational hinge engineering: modulating the conformational transformation of glutamate dehydrogenase for enhanced reductive amination activity towards non-natural substrates
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The active site is the common hotspot for rational and semi-rational enzyme activity engineering. However, the active site represents only a small portion of the whole enzyme. Identifying more hotspots other than the active site for enzyme activity engineering should aid in the development of biocatalysts with better catalytic performance. Glutamate dehydrogenases (GluDHs) are promising and environmentally benign biocatalysts for the synthesis of valuable chirall-amino acids by asymmetric reductive amination of α-keto acids. GluDHs contain an inter-domain hinge structure that facilitates dynamic reorientations of the domains relative to each other. Such hinge-bending conformational motions of GluDHs play an important role in regulating the catalytic activity. Thus, the hinge region represents a potential hotspot for catalytic activity engineering for GluDHs. Herein, we report semi-rational activity engineering of GluDHs with the hinge region as the hotspot. Mutants exhibiting significantly improved catalytic activity toward several non-natural substrates were identified and the highest activity increase reached 104-fold. Molecular dynamics simulations revealed that enhanced catalytic activity may arise from improving the open/closed conformational transformation efficiency of the protein with hinge engineering. In the batch production of three valuablel-amino acids, the mutants exhibited significantly improved catalytic efficiency, highlighting their industrial potential. Moreover, the catalytic activity of several active site tailored GluDHs was also increased by hinge engineering, indicating that hinge and active site engineering are compatible. The results show that the hinge region is a promising hotspot for activity engineering of GluDHs and provides a potent alternative for developing high-performance biocatalysts toward chirall-amino acid production.
- Liu, Yayun,Meng, Lijun,Wu, Jianping,Yang, Lirong,Yin, Xinjian,Zhou, Haisheng
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p. 3376 - 3386
(2020/06/09)
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- Scope and limitations of reductive amination catalyzed by half-sandwich iridium complexes under mild reaction conditions
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The conversion of aldehydes and ketones to 1° amines could be promoted by half-sandwich iridium complexes using ammonium formate as both the nitrogen and hydride source. To optimize this method for green chemical synthesis, we tested various carbonyl substrates in common polar solvents at physiological temperature (37 °C) and ambient pressure. We found that in methanol, excellent selectivity for the amine over alcohol/amide products could be achieved for a broad assortment of carbonyl-containing compounds. In aqueous media, selective reduction of carbonyls to 1° amines was achieved in the absence of acids. Unfortunately, at Ir catalyst concentrations of 1 mM in water, reductive amination efficiency dropped significantly, which suggest that this catalytic methodology might be not suitable for aqueous applications where very low catalyst concentration is required (e.g., inside living cells).
- Nguyen, Dat P.,Sladek, Rudolph N.,Do, Loi H.
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supporting information
(2020/07/15)
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- PERMANENTLY POLARIZED HYDROXYAPATITE, A PROCESS FOR ITS MANUFACTURE AND USES THEREOF
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The present invention relates to a permanently polarized hydroxyapatite and a composition or material comprising thereof. The present invention further relates to a process for obtaining a permanently polarized hydroxyapatite and to different uses of the permanently polarized hydroxyapatite or the composition or material comprising thereof.
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Paragraph 0191-0199
(2020/07/04)
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- Catalytic Production of Alanine from Waste Glycerol
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Chemical synthesis of amino acids directly from biomass feedstock is rare. Reported here is a one-step protocol to convert crude glycerol, from the biodiesel industry, into 43 % alanine over a Ru1Ni7/MgO catalyst. The multifunctional catalytic system promotes glycerol conversion into lactic acid, and then into alanine. X-ray absorption spectroscopy and scanning transmission electron microscopy revealed the existence of bimetallic RuNi species, whereas density-functional theory calculations suggested Ni-doped Ru substantially decreased the Ea of C?H bond dissociation of lactate alkoxide to form pyruvate, which is the rate-determining step. The catalytic route established in this work creates new opportunities for glycerol utilization and enriches the substrate scope of renewable feedstock to access value-added amino acids.
- Wang, Yunzhu,Furukawa, Shinya,Song, Song,He, Qian,Asakura, Hiroyuki,Yan, Ning
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supporting information
p. 2289 - 2293
(2020/01/08)
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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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- Robustness, Entrainment, and Hybridization in Dissipative Molecular Networks, and the Origin of Life
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How simple chemical reactions self-assembled into complex, robust networks at the origin of life is unknown. This general problem-self-assembly of dissipative molecular networks-is also important in understanding the growth of complexity from simplicity in molecular and biomolecular systems. Here, we describe how heterogeneity in the composition of a small network of oscillatory organic reactions can sustain (rather than stop) these oscillations, when homogeneity in their composition does not. Specifically, multiple reactants in an amide-forming network sustain oscillation when the environment (here, the space velocity) changes, while homogeneous networks-those with fewer reactants-do not. Remarkably, a mixture of two reactants of different structure-neither of which produces oscillations individually-oscillates when combined. These results demonstrate that molecular heterogeneity present in mixtures of reactants can promote rather than suppress complex behaviors.
- Cafferty, Brian J.,Wong, Albert S. Y.,Semenov, Sergey N.,Belding, Lee,Gmür, Samira,Huck, Wilhelm T. S.,Whitesides, George M.
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supporting information
p. 8289 - 8295
(2019/06/04)
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- Synthesis of Small Fluorescent Molecules and Evaluation of Photophysical Properties
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A series of aniline-based fluorophores were newly synthesized. To increase their fluorescence quantum yields, it was particularly important to substitute 3,3,3-trifluoroprop-1-enyl (TFPE) groups next to the amino group to benefit from an extended π-electron delocalization. Among these, 5-CN-2-TFPE-aniline was found to behave as an excellent fluorophore with a reasonable fluorescence quantum yield of 0.89 even in aqueous solution. l-Alanine peptide, a nonfluorescent analogue of 5-CN-2-TFPE-aniline, was synthesized and successfully employed as an enzyme probe to detect aminopeptidase N activity.
- Funabiki, Kazumasa,Hori, Kazushige,Ito, Kiyoshi,Karuo, Yukiko,Kawai, Kentaro,Miyanaga, Kanae,Ogawa, Futa,Omote, Masaaki,Saito, Yuki,Sato, Kazuyuki,Tani, Keita,Tarui, Atsushi,Yamada, Kengo,Yamazawa, Ryuji
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p. 1253 - 1258
(2020/02/04)
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- Krisynomycins, Imipenem Potentiators against Methicillin-Resistant Staphylococcus aureus, Produced by Streptomyces canus
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A reinvestigation of the acetone extract of the strain CA-091830 of Streptomyces canus, producer of the imipenem potentiator krisynomycin, resulted in the isolation of two additional analogues, krisynomycins B (1) and C (2), with different chlorination patterns. Genome sequencing of the strain followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster (BGC) of this cyclic nonribosomal peptide family. The planar structure of the new molecules was determined using HRMS, ESI-qTOF-MS/MS, and 1D and 2D NMR data. Their absolute configuration was proposed using a combination of Marfey's and bioinformatic BGC analyses. The krisynomycins displayed weak to negligible antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA), which was significantly enhanced when tested in combination with sublethal concentrations of imipenem. The halogenation pattern plays a key role in the antimicrobial activity and imipenem-potentiating effects of the compounds, with molecules having a higher number of chlorine atoms potentiating the effect of imipenem at lower doses.
- De La Cruz, Mercedes,Genilloud, Olga,González, Ignacio,Martín, Jesús,Oves-Costales, Daniel,Pérez-Bonilla, Mercedes,Reyes, Fernando,Vicente, Francisca
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p. 2597 - 2606
(2020/10/12)
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- Structure-guided engineering of: Pseudomonas dacunhae l-aspartate β-decarboxylase for l-homophenylalanine synthesis
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Structure-guided engineering of Pseudomonas dacunhael-aspartate β-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15400-fold improvement in specific activity reaching 216 mU mg-1, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.
- Zhang, Min,Hu, Pengfei,Zheng, Yu-Cong,Zeng, Bu-Bing,Chen, Qi,Zhang, Zhi-Jun,Xu, Jian-He
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p. 13876 - 13879
(2020/11/18)
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- Exploration of Transaminase Diversity for the Oxidative Conversion of Natural Amino Acids into 2-Ketoacids and High-Value Chemicals
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The use of 2-ketoacids is very common in feeds, food additives, and pharmaceuticals, and 2-ketoacids are valuable precursors for a plethora of chemically diverse compounds. Biocatalytic synthesis of 2-ketoacids starting from l-amino acids would be highly desirable because the substrates are readily available from biomass feedstock. Here, we report bioinformatic exploration of a series of aminotransferases (ATs) to achieve the desired conversion. Thermodynamic control was achieved by coupling an l-glutamate oxidation reaction in the cascade for the recycling of the amine acceptor. These enzymes were able to convert a majority of proteinogenic amino acids into the corresponding 2-ketoacids with high conversion (up to 99percent) and atom-efficiency. Furthermore, this enzyme cascade was extendable, and one-pot two-step processes were established for the synthesis of d-amino acids and N-methylated amino acids, achieving great overall conversion (up to 99percent) and high ee values (>99percent). These developed enzymatic methodologies offer convenient routes for utilizing amino acids as synthetic reagents.
- Chen, Yanchun,Cui, Xuexian,Cui, Yinglu,Li, Chuijian,Li, Ruifeng,Li, Tao,Sun, Jinyuan,Wu, Bian,Zhu, Tong
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p. 7950 - 7957
(2020/08/21)
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- Pagoamide A, a Cyclic Depsipeptide Isolated from a Cultured Marine Chlorophyte, Derbesia sp., Using MS/MS-Based Molecular Networking
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A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa, and pagoamide A was isolated using guidance by MS/MS-based molecular networking. Cultures were grown in a light- and temperature-controlled environment and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D and 2D NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey's analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.
- Cottrell, Garrison W.,Fang, Fang,Gerwick, Lena,Gerwick, William H.,Glukhov, Evgenia,Guan, Huashi,Kim, Hyunwoo,Leao, Tiago,Li, Yueying,Mao, Huanru Henry,Murray, Thomas F.,Pierce, Marsha L.,Yu, Hao-Bing,Zhang, Chen,Zhang, Yi
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supporting information
(2020/01/31)
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- Direct Synthesis of Free α-Amino Acids by Telescoping Three-Step Process from 1,2-Diols
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A practical telescoping three-step process for the syntheses of α-amino acids from the corresponding 1,2-diols has been developed. This process enables the direct synthesis of free α-amino acids without any protection/deprotection step. This method was also effective for the preparation of a 15N-labeled α-amino acid. 1,2-Diols bearing α,β-unsaturated ester moieties afforded bicyclic α-amino acids through intramolecular [3 + 2] cycloadditions. A preliminary study suggests that the resultant α-amino acids are resolvable by aminoacylases with almost complete selectivity.
- Inada, Haruki,Shibuya, Masatoshi,Yamamoto, Yoshihiko
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supporting information
p. 709 - 713
(2019/01/25)
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- Electrosynthesis of amino acids from biomass-derivable acids on titanium dioxide
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Seven amino acids were electrochemically synthesized from biomass-derivable α-keto acids and NH2OH with faradaic efficiencies (FEs) of 77-99% using an earth-Abundant TiO2 catalyst. Furthermore, we newly constructed a flow-Type electrochemical reactor, named a "polymer electrolyte amino acid electrosynthesis cell", and achieved continuous production of alanine with an FE of 77%.
- Fukushima, Takashi,Yamauchi, Miho
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supporting information
p. 14721 - 14724
(2019/12/24)
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- METHOD FOR PRODUCING alpha-AMINO ACID
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The present invention relates to a method for producing a specified α-amino acid, the method including allowing a specified α-amino acid amide and water to react with each other in the presence of a zirconium compound which contains zirconium and at least one metal element selected from the group consisting of lithium, nickel, copper, zinc, cesium, barium, hafnium, tantalum, cerium, and dysprosium.
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Paragraph 0157-0160
(2019/06/24)
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- Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids
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Two biocatalytic reactions, transamination catalyzed by transaminases and reductive amination catalyzed by amino acid dehydrogenases, can be used for asymmetric synthesis of optically pure unnatural amino acids. However, although transaminases show a great diversity and broad substrate spectrum, most transaminase reactions are reversible, while amino acid dehydrogenases catalyze reductive amination irreversibly but with strict substrate specificity. Accordingly, herein we developed a tri-enzyme one-pot reaction system to exploit the respective advantages of transaminases and amino acid dehydrogenases, while overcoming the disadvantages of each. In this work, representatives of all four subgroups of transaminases coupled with different amino acid dehydrogenases to produce five l- and four d- unnatural amino acid products, using ammonia and the co-enzyme NAD(P)H, which is regenerated by a robust alcohol dehydrogenase with 2-propanol as cheap cosubstrate. The complete conversion and high enantiopurity (ee > 99 %) of the products, demonstrated it as an ideal alternative for asymmetric synthesis of chiral amino acid compounds.
- Zhou, Haisheng,Meng, Lijun,Yin, Xinjian,Liu, Yayun,Xu, Gang,Wu, Jianping,Wu, Mianbin,Yang, Lirong
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p. 6470 - 6477
(2019/11/02)
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- ANTI-HUMAN PAPILLOMAVIRUS (HPV) ANTIGEN-BINDING PROTEINS AND METHODS OF USE THEREOF
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The present invention provides antigen-binding proteins that specifically bind to an HLA-displayed human papillomavirus (HPV) peptide, and therapeutic and diagnostic methods of using those binding proteins.
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- The identification and use of robust transaminases from a domestic drain metagenome
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Transaminases remain one of the most promising biocatalysts for use in chiral amine synthesis, however their industrial implementation has been hampered by their general instability towards, for example, high amine donor concentrations and organic solvent content. Herein we describe the identification, cloning and screening of 29 novel transaminases from a household drain metagenome. The most promising enzymes were fully characterised and the effects of pH, temperature, amine donor concentration and co-solvent determined. Several enzymes demonstrated good substrate tolerance as well as an unprecedented robustness for a wild-type transaminase. One enzyme in particular readily accepted IPA as an amine donor giving the same conversion with 2-50 equivalents, as well as being tolerant to a number of co-solvents, and operational in up to 50% DMSO-a characteristic as yet unobserved in a wild-type transaminase. This work highlights the value of using metagenomics for biocatalyst discovery from niche environments, and here has led to the identification of one of the most robust native transaminases described to date, with respect to IPA and DMSO tolerance.
- Leipold, Leona,Dobrijevic, Dragana,Jeffries, Jack W.E.,Bawn, Maria,Moody, Thomas S.,Ward, John M.,Hailes, Helen C.
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- DEHYDRATION AND AMINATION OF ALPHA-, BETA-DIHYDROXY CARBONYL COMPOUNDS TO ALPHA-AMINO ACIDS
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Processes are disclosed for synthesizing an α-amino acid or α-amino acid derivative, from a starting compound having a carbonyl functional group (C=O), with hydroxy-substituted carbon atoms at alpha (α) and beta (β) positions, relative to the carbonyl functional group. An α-, β-dihydroxy carboxylic acid or carboxylate is dehydrated to form a dicarbonyl intermediate by transformation of the α-hydroxy group to a second carbonyl group (adjacent a carbonyl group of the starting compound) and removal of the β-hydroxy group. This dicarbonyl intermediate is optionally cracked to form a second dicarbonyl intermediate having fewer carbon atoms but preserving the first and second carbonyl groups. Either or both of the dicarbonyl intermediate and the cracked dicarbonyl intermediate are then reductively aminated.
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Paragraph 55
(2019/11/04)
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- The roles of Ser-36, Asp-132 and Asp-201 in the reaction of Pseudomonas fluorescens Kynureninase
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Kynureninase from Pseudomonas fluorescens (Pfkynase)catalyzes the pyridoxal-5′-phosphate (PLP)dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and β-benzoyl-L-alanine, but not O-benzoyl-L-serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with β-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with β-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The 1H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold reduction of kcat and 30-fold reduction in kcat/Km with L-kynurenine, but very little effect on the reaction of O-benzoyl-L-serine. Thus, the rate-determining step in the reaction of S36A Pfkynase is the Cβ-Cγ bond cleavage. These results support the hypothesis that Ser-36 together with Tyr-226 is part of an oxyanion hole that polarizes the carbonyl of the substrate in the catalytic mechanism of Pfkynase.
- Phillips, Robert S.,Crocker, Mori,Lin, Richard,Idowu, O. Elijah,McCannon, David K.,Lima, Santiago
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p. 722 - 731
(2019/05/24)
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- Cold-induced aldimine bond cleavage by Tris in: Bacillus subtilis alanine racemase
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Pyridoxal 5′-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.
- Bernardo-García, Noelia,Sánchez-Murcia, Pedro A.,Espaillat, Akbar,Martínez-Caballero, Siseth,Cava, Felipe,Hermoso, Juan A.,Gago, Federico
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p. 4350 - 4358
(2019/05/10)
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- Chemical structure of cichorinotoxin, a cyclic lipodepsipeptide that is produced by Pseudomonas cichorii and causes varnish spots on lettuce
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Pseudomonas cichorii, which causes varnish spots on lettuce and seriously damages lettuce production during the summer season in the highland areas of Japan (e.g., Nagano and Iwate prefectures) was isolated. The structure of a toxin produced by this organism was analyzed based on the detailed evaluation of its 2D NMR and FABMS spectra, and this compound has not been reported previously. We propose the name cichorinotoxin for this toxin. In conjunction with the D or L configurations of each amino acid, which were determined by Marfey’s method, we propose the structure of cichorinotoxin to be as follows: 3-hydroxydecanoyl-(Z)-dhThr1-D-Pro2-D-Ala3-D-Ala4-D-Ala5-D-Val6-D-Ala7-(Z)-dhThr8-Ala9-Val10-D-Ile11-Ser12-Ala13-Val14-Ala15-Val16-(Z)-dhThr17-D-alloThr18-Ala19-L-Dab20-Ser21-Val22, and an ester linkage is present between D-alloThr18 and Val22 (dhThr: 2-aminobut-2-enoic acid; Dab: 2,4-diaminobutanoic acid). Thus, the toxin is a lipodepsipeptide with 22 amino acids. The mono- and tetraacetate derivatives and two alkaline hydrolysates, compounds A and B, were prepared. We discuss here the structure–activity relationships between the derivatives and their necrotic activities toward lettuce.
- Komatsu, Hidekazu,Shirakawa, Takashi,Uchiyama, Takeo,Hoshino, Tsutomu
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p. 299 - 309
(2019/02/20)
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- Isolation, structure elucidation and biological evaluation of lagunamide D: A new cytotoxic macrocyclic depsipeptide from marine cyanobacteria
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Lagunamide D, a new cytotoxic macrocyclic depsipeptide, was discovered from a collection of marine cyanobacteria from Loggerhead Key in the Dry Tortugas, Florida. An intramolecular ester exchange was observed, where the 26-membered macrocycle could contract to a 24-membered compound via acyl migration at the 1,3-diol unit, and the transformation product was named lagunamide D'. The planar structures of both compounds were elucidated using a combination of nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectroscopy (HRMS). The absolute configurations were determined on the basis of enantioselective analysis, modified Mosher's analysis, Kishi NMR database, and direct comparison with lagunamide A, a structure closely resembling lagunamide D. Lagunamides A and D displayed low-nanomolar antiproliferative activity against A549 human lung adenocarcinoma cells, while the structural transformation from the 26-membered lagunamide D macrocycle to the 24-membered ring structure for lagunamide D' led to a 9.6-fold decrease in activity. Lagunamide D also displayed potent activity in triggering apoptosis in a dose- and time-dependent manner. Further investigation on the mechanism of action of the lagunamide scaffold is needed to fully explore its therapeutic potential as an anticancer agent.
- Luo, Danmeng,Putra, Masteria Y.,Ye, Tao,Paul, Valerie J.,Luesch, Hendrik
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- Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone
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ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., R-analysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the R-analysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2×104-fold activity increase for 1-phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with >99% ee of a resulting (S)-amine. (Figure presented.).
- Kim, Hong-Gon,Han, Sang-Woo,Shin, Jong-Shik
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p. 2594 - 2606
(2019/05/15)
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