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János Rétey - One of the best experts on this subject based on the ideXlab platform.
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IDENTIFICATION OF ESSENTIAL AMINO ACIDS IN PHENYLALANINE AMMONIA-LYASE BY SITE-DIRECTED MUTAGENESIS
Biochemistry, 1997Co-Authors: Birgid Langer, Dagmar Rother, János RéteyAbstract:The postulated precursor of the prosthetic Dehydroalanine of phenylalanine ammonia-lyase (PAL), serine 202, was changed to cysteine by site-directed mutagenesis. After cloning and heterologous expr...
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Formation of the Michaelis complex without involvement of the prosthetic group Dehydroalanine of histidine ammonialyase
Bioorganic & Medicinal Chemistry Letters, 1997Co-Authors: Birgid Langer, Julia Starck, Martin Langer, János RéteyAbstract:Abstract The Dehydroalanine-less S143G mutant of histidine ammonia-lyase was constructed and used for kinetic measurements with 5′-nitro-histidine as a substrate. The natural substrate histidine turned out to be a competitive inhibitor of the mutant enzyme and exhibited a K i value which was similar to its K m value with the wild-type enzyme. Thus the Dehydroalanine prosthetic group does not play a role in formation of the Michaelis complex.
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Histidine ammonia-lyase mutant S143C is posttranslationally converted into fully active wild-type enzyme. Evidence for serine 143 to be the precursor of active site Dehydroalanine.
Biochemistry, 1994Co-Authors: Martin Langer, Annette Lieber, János RéteyAbstract:Histidase [histidine ammonia-lyase (HAL); EC 4.3.1.3] from Pseudomonas putida is a homotetramer and contains one catalytically essential Dehydroalanine residue per subunit. Since the mutant S143A was catalytically inert, it has been proposed that serine 143 is the precursor of the active site Dehydroalanine [Langer et al. (1994) Biochemistry 33, 6462-6467]. To further define the role of serine 143, we prepared the mutants S143T and S143C by site-directed mutagenesis. The threonine 143 mutant was neither catalytically active (< 0.01%) nor did it form with L-cysteine and oxygen a product absorbing at 340 nm. In contrast, the cysteine 143 mutant showed full catalytic activity and, after treatment with L-cysteine and oxygen, an increased absorbance at 340 nm similar to that of the wild-type enzyme. Also the kinetic constants (Km and Vmax) were identical with those of wild-type histidase. Titration with Ellman's reagent revealed that both wild-type and S143C mutant histidase contained seven thiol groups after exhaustive reduction. It must be concluded that posttranslational modification occurs with both serine 143 and cysteine 143 by elimination of water and hydrogen sulfide, respectively. In both cases Dehydroalanine is formed and the resulting histidases are indistinguishable. In contrast, the threonine 143 mutant is not processed to active enzyme.
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serine 202 is the putative precursor of the active site Dehydroalanine of phenylalanine ammonia lyase site directed mutagenesis studies on the enzyme from parsley petroselinum crispum l
FEBS Letters, 1994Co-Authors: Birgid Schuster, János RéteyAbstract:To investigate the possible role of serine as a precursor of Dehydroalanine at the active site of phenylalanine ammonia lyase, two serines, conserved in all known PAL and histidase sequences, were changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7.7 and the gene products were assayed for PAL activity. Mutant PALMutS209A showed the same catalytic property as wild-type PAL, whereas mutant PALMutS202A was devoid of catalytic activity, indicating that serine-202 is the most likely precursor of the active site Dehydroalanine.
Raymond F. Burk - One of the best experts on this subject based on the ideXlab platform.
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Loss of selenium from selenoproteins: Conversion of selenocysteine to Dehydroalanine in vitro
Journal of the American Society for Mass Spectrometry, 2003Co-Authors: Shuguang Ma, Kristina E Hill, Richard M Caprioli, Raymond F. BurkAbstract:Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to Dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to Dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-β-elimination of selenenic acid during sample processing.
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Loss of selenium from selenoproteins: Conversion of selenocysteine to Dehydroalanine in vitro
Journal of the American Society for Mass Spectrometry, 2003Co-Authors: Richard M Caprioli, Kristina E Hill, Raymond F. BurkAbstract:Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to Dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to Dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-beta-elimination of selenenic acid during sample processing.
Scott A. Mcluckey - One of the best experts on this subject based on the ideXlab platform.
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Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction
Analytical chemistry, 2019Co-Authors: David J. Foreman, Nicole C. Parsley, John T. Lawler, Uma K. Aryal, Leslie M. Hicks, Scott A. McluckeyAbstract:The gas-phase linearization of cyclotides via site-selective ring opening at Dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to...
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gas phase sequencing of cyclotides introduction of selective ring opening at Dehydroalanine via ion ion reaction
Analytical Chemistry, 2019Co-Authors: David J. Foreman, Nicole C. Parsley, John T. Lawler, Uma K. Aryal, Leslie M. Hicks, Scott A. McluckeyAbstract:The gas-phase linearization of cyclotides via site-selective ring opening at Dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to...
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The Dehydroalanine effect in the fragmentation of ions derived from polypeptides.
Journal of mass spectrometry : JMS, 2016Co-Authors: Alice L. Pilo, Zhou Peng, Scott A. McluckeyAbstract:Tandem mass spectrometry is a powerful approach for the analysis of peptides and proteins due to the primary structural information inherent in the observed products. The fragmentation of peptides and proteins depends heavily on the sequence and ion type of the species of interest. In this perspective special feature article, Scott McLuckey and co-authors show that peptides and proteins containing Dehydroalanine, a nonproteinogenic amino acid with an unsaturated side-chain, undergo enhanced cleavage of the N-Cα bond of the Dehydroalanine residue to generate c- and z-ions. Since these fragment ion types are not commonly observed upon activation of positively charged even-electron species, they can be used to identify Dehydroalanine residues and localize them within the peptide or protein chain. Scott McLuckey is Professor of Chemistry at Purdue University (West Lafayette, IN). His research interests are centered on gas-phase ion chemistry and instrumentation for organic and biological mass spectrometry.
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The Dehydroalanine effect in the fragmentation of ions derived from polypeptides.
Journal of mass spectrometry : JMS, 2016Co-Authors: Alice L. Pilo, Zhou Peng, Scott A. McluckeyAbstract:The fragmentation of peptides and proteins upon collision-induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing Dehydroalanine, a non-proteinogenic amino acid with an unsaturated side-chain, undergo enhanced cleavage of the N-Cα bond of the Dehydroalanine residue to generate c- and z-ions. Because these fragment ion types are not commonly observed upon activation of positively charged even-electron species, they can be used to identify Dehydroalanine residues and localize them within the peptide or protein chain. While Dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S-alkyl cysteine residues generate Dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates Dehydroalanine. Furthermore, we show that gas-phase ion/ion reactions can be used to facilitate the generation of Dehydroalanine residues via, for example, oxidation of S-alkyl cysteine residues and conversion of multiply-protonated peptides to radical cations. In the latter case, loss of radical side-chains to generate Dehydroalanine from some amino acids gives rise to the possibility for residue-specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.
Richard M Caprioli - One of the best experts on this subject based on the ideXlab platform.
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Loss of selenium from selenoproteins: Conversion of selenocysteine to Dehydroalanine in vitro
Journal of the American Society for Mass Spectrometry, 2003Co-Authors: Shuguang Ma, Kristina E Hill, Richard M Caprioli, Raymond F. BurkAbstract:Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to Dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to Dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-β-elimination of selenenic acid during sample processing.
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Loss of selenium from selenoproteins: Conversion of selenocysteine to Dehydroalanine in vitro
Journal of the American Society for Mass Spectrometry, 2003Co-Authors: Richard M Caprioli, Kristina E Hill, Raymond F. BurkAbstract:Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to Dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to Dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-beta-elimination of selenenic acid during sample processing.
Paula M. T. Ferreira - One of the best experts on this subject based on the ideXlab platform.
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synthesis of fluorescent alanines by a rhodium catalysed conjugate addition of arylboronic acids to Dehydroalanine derivatives
European Journal of Organic Chemistry, 2013Co-Authors: Paula M. T. Ferreira, Luis S Monteiro, Goreti Pereira, Elisabete M S Castanheira, Christopher G FrostAbstract:Several β-arylalanine derivatives containing fluorescent groups were prepared in good yields using a rhodium-catalysed conjugate addition of arylboronic acids to N,N-diprotected and N-monoprotected Dehydroalanines. The best conditions for these reactions required the use of an excess of arylboronic acid (4 equiv.), [Rh(COD)]BF as catalyst, and CsF as base in dioxane/HO (10:1) at 110 °C. These conditions were also applied to several dipeptides with Dehydroalanine residues. The photophysical properties of some of the β-arylalanines were studied in three solvents with different polarities. Due to the absence of the α,β-double bond, the absorption and fluorescence emission of the new compounds are dominated by the photophysical properties of the polycyclic aromatic fluorophores (naphthalene, phenanthrene, and pyrene). Considering the relatively high fluorescence quantum yield of these compounds, some of them may be useful as fluorescent markers for peptides and proteins. Fluorescent β-arylalanine derivatives were prepared in good yields using a rhodium-catalysed conjugate addition of arylboronic acids to N-protected Dehydroalanines. The photophysical properties of some of the β-arylalanines were studied in solvents of different polarities. Considering the high fluorescence quantum yield of these compounds, some of them may be useful as fluorescent markers.
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Synthesis of fluorescent alanines by a rhodium-catalysed conjugate addition of arylboronic acids to Dehydroalanine derivatives
European Journal of Organic Chemistry, 2012Co-Authors: Paula M. T. Ferreira, Luis S Monteiro, Goreti Pereira, Elisabete M S Castanheira, Christopher G FrostAbstract:Several arylalanine derivatives containing fluorescent groups were prepared in good yields using a rhodium catalysed conjugate addition of arylboronic acids to N,N-diprotected and N-monoprotected Dehydroalanines. The best conditions for these reactions require the use of an excess of aryl boronic acid (4 equiv.), [Rh(COD)2]BF4 as catalyst and CsF as base in dioxane:H2O (10:1) at 110 ºC. These conditions were also applied to several dipeptides with Dehydroalanine residues. The photophysical properties of some of the arylalanines were studied in three solvents of different polarity. Due to the absence of the, double bond, the absorption and fluorescence emission of the new compounds are dominated by the photophysical properties of the polycyclic aromatic fluorophores (naphthalene, phenanthrene and pyrene). Considering the relatively high fluorescence quantum yield of these compounds, some of them may be useful as fluorescent markers for peptides and proteins.Thanks are due to the Foundation for Science and Technology (FCT, Portugal), Quadro de Referencia Estrategico Nacional (QREN), and Fundo Europeu de Desenvolvimento Regional/Uniao Europeia (FEDER/EU) for financial support through the research centers, CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)] and CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-022711)], and project PTDC/QUI/81238/2006 (cofinanced by Fundo Europeu de Desenvolvimento Regional/Programa Operacional Fatores de Competitividade (FEDER/COMPETE), ref. FCOMP-01-0124-FEDER-007467). G. P. acknowledges her PhD grant from Fundacao para a Ciencia e a Tecnologia (FCT), Programa Operacional Potencial Humano/Quadro de Referencia Estrategico Nacional (POPH-QREN), Fundo Social Europeu (FSE) (SFRH/BD/38766/2007)
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Synthesis and Reactivity of β‐Bromo‐β‐Substituted Dehydroalanines
European Journal of Organic Chemistry, 2006Co-Authors: Paula M. T. Ferreira, Luis S MonteiroAbstract:The methyl ester of N-tert-butoxycarbonyl-(Z)-β-bromo-β-(1,2,4-triazol-1-yl)Dehydroalanine was prepared by treatment of the methyl ester of N-tert-butoxycarbonyl-(E)-β-(1,2,4-triazol-1-yl)Dehydroalanine with N-bromosuccinimide (NBS), followed by Et3N. The reactivities of this compound and of our previously synthesized methyl ester of N-tert-butoxycarbonyl-β,β-dibromoDehydroalanine towards several nucleophiles were studied, and it was found that these compounds react with oxygen nucleophiles to give the corresponding α-alkoxy-β,β-disubstituted alanines. Addition to the α-carbon atom also occurred when the β,β-dibromoDehydroalanine derivative was treated with primary amines, giving α-amino-β,β-dibromoalanines. Treatment of the β-bromo-β-(1,2,4-triazol-1-yl)Dehydroalanine derivative with amines gave α-(alkylamino)-β-(alkylimino)alanines in high yields. These iminoalanines afforded α-aminoglycines when treated with silica in dichloromethane. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
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Synthesis and intramolecular cyclization of novel β,β-bis-(benzo[b]thienyl)Dehydroalanine derivatives
Tetrahedron Letters, 2003Co-Authors: Ana S. Abreu, Natália O. Silva, Paula M. T. Ferreira, Maria João R. P. QueirozAbstract:Abstract The methyl ester of tert -butyloxycarbonyl-β,β-dibromoDehydroalanine was obtained in a one-pot procedure from bis-( N - tert -butyloxycarbonyl)Dehydroalanine. The former was reacted with several boronic benzo[ b ]thiophene acids under Suzuki cross coupling conditions, to give new β,β-bis-(benzo[ b ]thienyl)Dehydroalanines in high yields. These compounds were cyclized to pyrrole derivatives by treatment with Pd(OAc) 2 and Cu(OAc) 2 in DMF.
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New β,β-Bis(benzo[b]thienyl)Dehydroalanine Derivatives: Synthesis and Cyclization
European Journal of Organic Chemistry, 2003Co-Authors: Ana S. Abreu, Natália O. Silva, Paula M. T. Ferreira, Maria João R. P. Queiroz, Mariano VenanziAbstract:The methyl ester of β,β-dibromo-N-(tert-butoxycarbonyl)Dehydroalanine was synthesized by treating the methyl ester of N,N-bis(tert-butoxycarbonyl)Dehydroalanine with trifluoroacetic acid, N-bromosuccinimide and triethylamine. This compound was then used in Suzuki cross-coupling reactions with several (benzo[b]thienyl)boronic acids to give the corresponding β,β-bis(benzo[b]thienyl)Dehydroalanine derivatives in good to high yields (55−90 %). After several experiments, the best conditions were shown to be: (benzo[b]thienyl)boronic acid (5 equiv.), [Pd(PPh)2Cl2] (20 mol %), Na2CO3 (4 equiv.) in DME/H2O (10:1). The Suzuki cross-coupling products were treated with Pd(OAc)2 and Cu(OAc)2 in DMF at 160 °C to give the (benzo[b]thienyl)pyrroles in moderate to good yields (25−62 %). Other attempts were carried out using only Cu(OAc)2, the thienylpyrroles being, in some cases, isolated in lower yields. Preliminary fluorescence studies show that the (benzo[b]thienyl)pyrroles can be used as biomarkers. All of these compounds are non-proteinogenic amino acids that can have biological activity or can be used in conformational studies in order to establish structure-activity relationships. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)