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Hiroaki Suga - One of the best experts on this subject based on the ideXlab platform.
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tRNA engineering for manipulating genetic code
RNA biology, 2017Co-Authors: Takayuki Katoh, Yoshihiko Iwane, Hiroaki SugaAbstract:In ribosomal translation, only 20 kinds of Proteinogenic Amino Acids (pAAs), namely 19 l-Amino Acids and glycine, are exclusively incorporated into polypeptide chain. To overcome this limitation, various methods to introduce non-Proteinogenic Amino Acids (npAAs) other than the 20 pAAs have been developed to date. However, the repertoire of Amino Acids that can be simultaneously introduced is still limited. Moreover, the efficiency of npAA incorporation is not always sufficient depending on their structures. Fidelity of translation is sometimes low due to misincorporation of competing pAAs and/or undesired translation termination. Here, we provide an overview of efforts to solve these issues, focusing on the engineering of tRNAs.
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Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons
Chembiochem : a European journal of chemical biology, 2016Co-Authors: Ki Baek Lee, Hiroaki Suga, Dong-myung Kim, Chen Yuan Hou, Chae‐eun Kim, Taek Jin KangAbstract:The genetic code in most organisms codes for 20 Proteinogenic Amino Acids or translation stop. In order to encode more than 20 Amino Acids in the coding system, one of stop codons is usually reprogrammed to encode a non-Proteinogenic Amino acid. Although this approach works, usually only one Amino acid is added to the Amino acid repertoire. In this study, we incorporated non-Proteinogenic Amino Acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA(Arg) in a cell-free protein synthesis system by using a tRNA(Arg) -specific tRNase, colicin D. Then by supplementing the system with tRNACCU , the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 Proteinogenic Amino Acids.
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Expanding the Amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes
Nature Chemistry, 2016Co-Authors: Yoshihiko Iwane, Azusa Hitomi, Hiroshi Murakami, Takayuki Katoh, Yuki Goto, Hiroaki SugaAbstract:The repertoire of Amino Acids available for ribosomal peptide synthesis is limited by the genetic code. Now, a method to reduce the redundancy of codons has been developed based on the artificial division of codon boxes. This method enables non-Proteinogenic Amino Acids to be included in peptides without sacrificing Proteinogenic ones. In ribosomal polypeptide synthesis the library of Amino acid building blocks is limited by the manner in which codons are used. Of the Proteinogenic Amino Acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-Proteinogenic Amino Acids and thereby expand the library of genetically encoded Amino Acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNA_SNN (tRNA_SNN) (S = G or C), assigning the initiator and 20 elongator Amino Acids. Reassignment of three redundant codons was achieved by replacing redundant tRNA_SNNs with tRNA_SNNs pre-charged with non-Proteinogenic Amino Acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 Proteinogenic and three non-Proteinogenic Amino Acids, and a 14-mer macrocyclic peptide that contains more than four non-Proteinogenic Amino Acids.
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Expanding the Amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes
Nature chemistry, 2016Co-Authors: Yoshihiko Iwane, Azusa Hitomi, Hiroshi Murakami, Takayuki Katoh, Yuki Goto, Hiroaki SugaAbstract:In ribosomal polypeptide synthesis the library of Amino acid building blocks is limited by the manner in which codons are used. Of the Proteinogenic Amino Acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-Proteinogenic Amino Acids and thereby expand the library of genetically encoded Amino Acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator Amino Acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-Proteinogenic Amino Acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 Proteinogenic and three non-Proteinogenic Amino Acids, and a 14-mer macrocyclic peptide that contains more than four non-Proteinogenic Amino Acids.
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Discovering functional, non-Proteinogenic Amino acid containing, peptides using genetic code reprogramming.
Organic & biomolecular chemistry, 2015Co-Authors: Joseph M. Rogers, Hiroaki SugaAbstract:The protein synthesis machinery of the cell, the ribosome and associated factors, is able to accurately follow the canonical genetic code, that which maps RNA sequence to protein sequence, to assemble functional proteins from the twenty or so Proteinogenic Amino Acids. A number of innovative methods have arisen to take advantage of this accurate, and efficient, machinery to direct the assembly of non-Proteinogenic Amino Acids. We review and compare these routes to ‘reprogram the genetic code’ including in vitro translation, engineered Aminoacyl tRNA synthetases, and RNA ‘flexizymes’. These studies show that the ribosome is highly tolerant of unnatural Amino Acids, with hundreds of unusual substrates of varying structure and chemistries being incorporated into protein chains. We also discuss how these methods have been coupled to selection techniques, such as phage display and mRNA display, opening up an exciting new avenue for the production of proteins and peptides with properties and functions beyond that which is possible using proteins composed entirely of the Proteinogenic Amino Acids.
Hans Bruckner - One of the best experts on this subject based on the ideXlab platform.
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determination and stereochemistry of Proteinogenic and non Proteinogenic Amino Acids in saudi arabian date fruits
Amino Acids, 2014Co-Authors: Hatem Salama Mohamed Ali, Omar Amin Alhaj, Abdulrahman S Alkhalifa, Hans BrucknerAbstract:Whereas an abundance of literature is available on the occurrence of common Proteinogenic Amino Acids (AAs) in edible fruits of the date palm (Phoenix dactylifera L.), recent reports on non-Proteinogenic (non-coded) AAs and Amino components are scarce. With emphasis on these components we have analyzed total hydrolysates of twelve cultivars of date fruits using automated ion-exchange chromatography, HPLC employing a fluorescent Aminoquinolyl label, and GC–MS of total hydrolysates using the chiral stationary phases Chirasil®-L-Val and Lipodex® E. Besides common Proteinogenic AAs, relatively large amounts of the following non-Proteinogenic Amino Acids were detected: (2S,5R)-5-hydroxypipecolic acid (1.4–4.0 g/kg dry matter, DM), 1-Aminocyclopropane-1-carboxylic acid (1.3–2.6 g/kg DM), γ-Amino-n-butyric acid (0.5–1.2 g/kg DM), (2S,4R)-4-hydroxyproline (130–230 mg/kg DM), l-pipecolic acid (40–140 mg/kg DM), and 2-Aminoethanol (40–160 mg/kg DM) as well as low or trace amounts (<70 mg/kg DM) of l-ornithine, 5-hydroxylysine, β-alanine, and in some samples (<20 mg/kg DM) of (S)-β-Aminoisobutyric acid and (<10 mg/kg DM) l-allo-isoleucine. In one date fruit, traces of α-Aminoadipic acid could be determined. Enantiomeric analysis of 6 M DCl/D2O hydrolysates of AAs using chiral capillary gas chromatography–mass spectrometry revealed the presence of very low amounts of d-Ala, d-Asp, d-Glu, d-Ser and d-Phe (1.2–0.4 %, relative to the corresponding l-enantiomers), besides traces (0.2–1 %) of other d-AAs. The possible relevance of non-Proteinogenic Amino Acids in date fruits is briefly addressed.
Hatem Salama Mohamed Ali - One of the best experts on this subject based on the ideXlab platform.
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Determination and stereochemistry of Proteinogenic and non-Proteinogenic Amino Acids in Saudi Arabian date fruits
Amino Acids, 2014Co-Authors: Hatem Salama Mohamed Ali, Omar Amin Alhaj, Abdulrahman Saleh Al-Khalifa, Hans BrücknerAbstract:Whereas an abundance of literature is available on the occurrence of common Proteinogenic Amino Acids (AAs) in edible fruits of the date palm (Phoenix dactylifera L.), recent reports on non-Proteinogenic (non-coded) AAs and Amino components are scarce. With emphasis on these components we have analyzed total hydrolysates of twelve cultivars of date fruits using automated ion-exchange chromatography, HPLC employing a fluorescent Aminoquinolyl label, and GC–MS of total hydrolysates using the chiral stationary phases Chirasil®-L-Val and Lipodex® E. Besides common Proteinogenic AAs, relatively large amounts of the following non-Proteinogenic Amino Acids were detected: (2S,5R)-5-hydroxypipecolic acid (1.4–4.0 g/kg dry matter, DM), 1-Aminocyclopropane-1-carboxylic acid (1.3–2.6 g/kg DM), γ-Amino-n-butyric acid (0.5–1.2 g/kg DM), (2S,4R)-4-hydroxyproline (130–230 mg/kg DM), l-pipecolic acid (40–140 mg/kg DM), and 2-Aminoethanol (40–160 mg/kg DM) as well as low or trace amounts (
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determination and stereochemistry of Proteinogenic and non Proteinogenic Amino Acids in saudi arabian date fruits
Amino Acids, 2014Co-Authors: Hatem Salama Mohamed Ali, Omar Amin Alhaj, Abdulrahman S Alkhalifa, Hans BrucknerAbstract:Whereas an abundance of literature is available on the occurrence of common Proteinogenic Amino Acids (AAs) in edible fruits of the date palm (Phoenix dactylifera L.), recent reports on non-Proteinogenic (non-coded) AAs and Amino components are scarce. With emphasis on these components we have analyzed total hydrolysates of twelve cultivars of date fruits using automated ion-exchange chromatography, HPLC employing a fluorescent Aminoquinolyl label, and GC–MS of total hydrolysates using the chiral stationary phases Chirasil®-L-Val and Lipodex® E. Besides common Proteinogenic AAs, relatively large amounts of the following non-Proteinogenic Amino Acids were detected: (2S,5R)-5-hydroxypipecolic acid (1.4–4.0 g/kg dry matter, DM), 1-Aminocyclopropane-1-carboxylic acid (1.3–2.6 g/kg DM), γ-Amino-n-butyric acid (0.5–1.2 g/kg DM), (2S,4R)-4-hydroxyproline (130–230 mg/kg DM), l-pipecolic acid (40–140 mg/kg DM), and 2-Aminoethanol (40–160 mg/kg DM) as well as low or trace amounts (<70 mg/kg DM) of l-ornithine, 5-hydroxylysine, β-alanine, and in some samples (<20 mg/kg DM) of (S)-β-Aminoisobutyric acid and (<10 mg/kg DM) l-allo-isoleucine. In one date fruit, traces of α-Aminoadipic acid could be determined. Enantiomeric analysis of 6 M DCl/D2O hydrolysates of AAs using chiral capillary gas chromatography–mass spectrometry revealed the presence of very low amounts of d-Ala, d-Asp, d-Glu, d-Ser and d-Phe (1.2–0.4 %, relative to the corresponding l-enantiomers), besides traces (0.2–1 %) of other d-AAs. The possible relevance of non-Proteinogenic Amino Acids in date fruits is briefly addressed.
Wolfgang Lindner - One of the best experts on this subject based on the ideXlab platform.
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a comparative study of enantioseparations of nα fmoc Proteinogenic Amino Acids on quinine based zwitterionic and anion exchanger type chiral stationary phases under hydro organic liquid and subcritical fluid chromatographic conditions
Molecules, 2016Co-Authors: Gyula Lajko, Gabor Toth, Ferenc Fulop, Wolfgang Lindner, Antal Peter, Nora Grecso, Istvan IliszAbstract:The focus of this contribution is a comparative investigation of enantioseparations of 19 Nα-Fmoc Proteinogenic Amino Acids on Quinine-based zwitterionic and anion-exchanger type chiral stationary phases employing hydro-organic and polar-ionic liquid and subcritical fluid chromatographic conditions. Effects of mobile phase composition (including additives, e.g., water, basis and Acids) and nature of chiral selectors on the chromatographic performances were studied at different chromatographic modes. Thermodynamic parameters of the temperature dependent enantioseparation results were calculated in the temperature range 5–50 °C applying plots of lnα versus 1/T. The differences in standard enthalpy and standard entropy for a given pair of enantiomers were calculated and served as a basis for comparisons. Elution sequence in all cases was determined, where a general rule could be observed, both in liquid and subcritical fluid chromatographic mode the d-enantiomers eluted before the L ones. In both modes, the principles of ion exchange chromatography apply.
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application of cinchona alkaloid based zwitterionic chiral stationary phases in supercritical fluid chromatography for the enantioseparation of nα protected Proteinogenic Amino Acids
Journal of Chromatography A, 2015Co-Authors: Gyula Lajko, Istvan Ilisz, Gabor Toth, Ferenc Fulop, Wolfgang Lindner, Antal PeterAbstract:Abstract Stereoselective supercritical fluid chromatographic separations of the enantiomers of a large set of Nα-Fmoc Proteinogenic Amino Acids were carried out on the recently developed Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+)™ and ZWIX(−)™ with protic solvents as co-solvents. The effects of the mobile phase composition, the natures and concentrations of the acid or base additives, the co- and counter-ions and temperature on the separations were investigated. The retention time in most cases slightly increased, while the separation factor decreased with increasing temperature. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van’t Hoff plots derived from the ln α vs 1/T curves in the studied temperature range (20–60 °C). The values of the thermodynamic parameters depended on the natures of the selectors and the structures of the analytes. On both ZWIX(+)™ and ZWIX(−)™ columns, enthalpically-driven separations were observed. The elution sequence was determined in all cases and was observed to be opposite on ZWIX(+)™ and ZWIX(−)™ which acted for the presented applications as chiral anion exchanger.
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Methoxyquinoline labeling--a new strategy for the enantioseparation of all chiral Proteinogenic Amino Acids in 1-dimensional liquid chromatography using fluorescence and tandem mass spectrometric detection.
Journal of chromatography. A, 2012Co-Authors: Roland Reischl, Wolfgang LindnerAbstract:Abstract The determination of trace amounts of d -Amino Acids ( d -AAs) even in tissue samples of higher developed animals, mammals and humans has opened a wide field of biological questions to be investigated. d -Ala, d -Asp and d -Ser have already been identified to exhibit key functions in cellular regulation processes [1] , [2] , [3] , [4] . The abundance of trace amounts of these and also of other d -AAs in various biological fluids and in tissue samples is still being investigated. We herein present a facile derivatization method for Amino Acids using 6-methoxyquinoline-4-carboxylic acid-succinimide ester (MQ-OSu) to yield the corresponding stable N-acyl-Amino Acids (MQ-AAs). Labeling with the MQ tag supports the enantioseparation of all 19 chiral Proteinogenic Amino Acids on anion exchanger type chiral stationary phases, introduces fluorescence activity and particularly promotes sensitive electrospray tandem mass spectrometric detection. Limit of detection values (LOD) for MQ- l -Ala in water were 1.25 μmol L−1 with fluorescence detection and 0.015 μmol L−1 with MS in selected reaction monitoring (SRM) mode. The applicability of this method for the analysis of MQ- d -AAs in biological fluids has been demonstrated.
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chemoselective and enantioselective analysis of Proteinogenic Amino Acids utilizing n derivatization and 1 d enantioselective anion exchange chromatography in combination with tandem mass spectrometric detection
Journal of Chromatography A, 2011Co-Authors: Roland Reischl, Lucie Hartmanova, Marina M Carrozzo, Monika Huszar, Peter Fruhauf, Wolfgang LindnerAbstract:Abstract d -Amino acid analysis in biological samples still poses a challenge to analytical chemists. In higher developed species trace amounts of d -Amino Acids have to be detected in vast excesses of the corresponding l- enantiomers. This method utilizes an easy-to-carry-out derivatization step on the Amino group with an iron ferrocenyl propionate hydroxy succinimide ester followed by one-dimensional enantioselective anion exchange chromatography with cinchona alkaloid based chiral stationary phases (CSPs). MS detection is carried out in the highly sensitive SRM (selected reaction monitoring) mode, which allows a chemoselective differentiation of Amino acid derivatives as well as their enantioselective separation in one step. Application of this method allows LOD (limits of detection) in the low μmol L−1 range and baseline enantioseparation for all Proteinogenic Amino Acids except for Pro, Arg and His. The d- enantiomers of isomeric Leu and Ile were separated chromatographically and pose an example for the complementary selectivities of LC and MS. A successful application of this procedure to unprocessed human urine indicated the eligibility to analyse biological samples.
Matthias Laska - One of the best experts on this subject based on the ideXlab platform.
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Gustatory responsiveness to the 20 Proteinogenic Amino Acids in the spider monkey (Ateles geoffroyi)
Physiology & Behavior, 2014Co-Authors: Jenny Larsson, Laura Teresa Hernandez Salazar, Anna Maitz, Matthias LaskaAbstract:The gustatory responsiveness of four adult spider monkeys to the 20 Proteinogenic Amino Acids was assessed in two-bottle preference tests of brief duration (1 min). We found that Ateles geoffroyi responded with significant preferences for seven Amino Acids (glycine, l-proline, l-alanine, l-serine, l-glutamic acid, l-aspartic acid, and l-lysine) when presented at a concentration of 100 mM and/or 200 mM and tested against water. At the same concentrations, the animals significantly rejected five Amino Acids (l-tryptophan, l-tyrosine, l-valine, l-cysteine, and l-isoleucine) and were indifferent to the remaining tastants. Further, the results show that the spider monkeys discriminated concentrations as low as 0.2 mM l-lysine, 2 mM l-glutamic acid, 10 mM l-proline, 20 mM l-valine, 40 mM glycine, l-serine, and l-aspartic acid, and 80 mM l-alanine from the alternative stimulus, with individual animals even scoring lower threshold values. A comparison between the taste qualities of the Proteinogenic Amino Acids as described by humans and the preferences and aversions observed in the spider monkeys suggests a fairly high degree of agreement in the taste quality perception of these tastants between the two species. A comparison between the taste preference thresholds obtained with the spider monkeys and taste detection thresholds reported in human subjects suggests that the taste sensitivity of A. geoffroyi for the Amino Acids tested here might match that of Homo sapiens. The results support the assumption that the taste responses of spider monkeys to Proteinogenic Amino Acids might reflect an evolutionary adaptation to their frugivorous and thus protein-poor diet.
