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Paul F. Fitzpatrick - One of the best experts on this subject based on the ideXlab platform.
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Structure of the flavoprotein Tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants.
Biochemistry, 2013Co-Authors: Helena Gaweska, Alexander B. Taylor, P. John Hart, Paul F. FitzpatrickAbstract:The flavoprotein Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of Tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from Pseudomonas savastanoi has been determined by X-ray diffraction methods to a resolution of 1.95 A. The overall structure of the protein shows that it has the same fold as members of the monoamine oxidase family of flavoproteins, with the greatest similarities to the l-amino acid oxidases. The location of bound indole-3-acetamide in the active site allows identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the kcat and kcat/KTrp values by 60000- and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, ...
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Mechanistic studies of the flavoenzyme Tryptophan 2-monooxygenase: deuterium and 15N kinetic isotope effects on alanine oxidation by an L-amino acid oxidase.
Biochemistry, 2006Co-Authors: Erik C. Ralph, Mark Anderson, W. Wallace Cleland, Paul F. FitzpatrickAbstract:Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of l-Tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically, the enzyme is a member of the family of l-amino acid oxidases. Deuterium and 15N kinetic isotope effects were used to probe the chemical mechanism of l-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH independent over the pH range 6.5-10, with an average value of 6.0 +/- 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 +/- 0.9; no intermediate flavin species were observed during flavin reduction. The kcat/Kala value was 1.0145 +/- 0.0007 at pH 8. NMR analyses gave an equilibrium 15N isotope effect for deprotonation of the alanine amino group of 1.0233 +/- 0.0004, allowing calculation of the 15N isotope effect on the CH bond cleavage step of 0.9917 +/- 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.
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Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family
Biochemistry, 2003Co-Authors: Pablo Sobrado, Paul F. FitzpatrickAbstract:The flavoprotein Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of Tryptophan to indoleacetamide. We have previously identified Tryptophan 2-monooxygenase as a homologue of L-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402, 24-30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of Tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A Tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the K d value for Tryptophan or the K i value for the competitive inhibitor indoleacetamide, and increases the K i value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5- and 2-fold, respectively, and increases the K d value for Tryptophan and the K i value for indolepyruvate by 31- and 17-fold, respectively, while having an only 2-fold effect on the K i value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with Tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/K t r p values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis.
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Identification of Tyr413 as an active site residue in the flavoprotein Tryptophan 2-monooxygenase and analysis of its contribution to catalysis.
Biochemistry, 2003Co-Authors: Pablo Sobrado, Paul F. FitzpatrickAbstract:The flavoenzyme Tryptophan 2-monooxygenase catalyzes the oxidation of Tryptophan to indoleacetamide, carbon dioxide, and water. The enzyme is a homologue of l-amino acid oxidase. In the structure of l-amino acid oxidase complexed with aminobenzoate, Tyr372 hydrogen bonds with the carboxylate of the inhibitor in the active site. All 10 conserved tyrosine residues in Tryptophan 2-monooxygenase were mutated to phenylalanine; steady state kinetic characterization of the purified proteins identified Tyr413 as the residue homologous to Tyr372 of l-amino acid oxidase. Y413F and Y413A Tryptophan 2-monooxygenase were characterized more completely with Tryptophan as the substrate to probe the contribution of this residue to catalysis. Mutation of Tyr413 to phenylalanine results in a decrease in the value of the first-order rate constant for reduction of 35-fold and a decrease in the rate constant for oxidation of 11-fold. Mutation to alanine decreases the rate constant for reduction by 200-fold and that for oxidation by 33-fold. Both mutations increase the K(d) value for Tryptophan and the K(i) values for the competitive inhibitors indoleacetamide and indole pyruvate by 5-10-fold. Both mutations convert the enzyme to an oxidase, in that the products of the catalytic reactions of both are indolepyruvate and hydrogen peroxide. The V/K(trp)-pH profiles for the Tyr413 mutant enzymes no longer show the pK(a) value of 9.9 seen in that for the wild-type enzyme, allowing identification of Tyr413 as the active site residue in the wild-type enzyme which must be protonated for catalysis. Substitution of Tyr413 abolishes the formation of the long wavelength charge transfer species observed in the wild-type enzyme. The data are consistent with the main role of Tyr413 being to maintain the correct orientation of Tryptophan for effective hydride transfer and imino acid decarboxylation.
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Analysis of the roles of amino acid residues in the flavoprotein Tryptophan 2-monooxygenase modified by 2-oxo-3-pentynoate: characterization of His338, Cys339, and Cys511 mutant enzymes
Archives of biochemistry and biophysics, 2002Co-Authors: Pablo Sobrado, Paul F. FitzpatrickAbstract:The flavoprotein Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of Tryptophan to indoleacetamide. His338, Cys339, and Cys511 of the Pseudomonas savastanoi enzyme were previously identified as possible active-site residues by modification with 2-oxo-3-pentynoate ([G. Gadda, L.J. Dangott, W.H. Johnson Jr., C.P. Whitman, P.F. Fitzpatrick, Biochemistry 38 (1999) 5822-5828]). The H338N, C339A, and C511S enzymes have been characterized to determine the roles of these residues in catalysis. The steady-state kinetic parameters with both Tryptophan and methionine decrease only slightly in the case of the H338N and C339A enzymes; the decrease in activity is greater for the C511S enzyme. Only in the case of the C511S enzyme do deuterium kinetic isotope effects on kinetic parameters indicate a significant change in catalytic rates. The structural bases for the effects of the mutations can be interpreted by identification of L-amino acid oxidase and Tryptophan monooxygenase as homologous proteins.
Roger Genet - One of the best experts on this subject based on the ideXlab platform.
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Diastereoselective deuteration of (Z)-α,β-dehydroTryptophanyl-containing biological peptides controlled by chiral rhodium catalysts
Tetrahedron Letters, 1998Co-Authors: Akli Hammadi, André Ménez, Gilles Meunier, Roger GenetAbstract:Abstract The regio- and stereoselective labelling ( 2 H, 3 H) of Tryptophanyl containing biological peptides catalysed by chiral rhodium complexes is described. The Δ Z Trp-residue is directly produced in the peptide by a catalytic process, involving the ( S )-Tryptophan 2′,3′-oxydase from Chromobacterium violaceum . Two biological dehydropentapeptides, pGlu-His-Δ Z Trp-Ser-Tyr and Boc-βAla-Δ Z Trp-Met-Asp-Phe-NH 2 , have been thus synthesized. Asymmetric deuteration of these compounds was then investigated in the presence of various rhodium catalysts. High stereocontrol was achieved by suitable choice of the chiral ligands. This strategy offers new possibilities for the asymmetric synthesis of labelled ( 2 H, 3 H) peptides without ultimate deprotection step.
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Asymmetric deuteration of N-acetyl-(Z)-α,β-dehydroTryptophan-(L)-phenylalanine methyl ester produced by (L)-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum. A new route for stereospecific labelling of peptides
Tetrahedron, 1997Co-Authors: Akli Hammadi, André Ménez, Roger GenetAbstract:Abstract A novel approach to the synthesis of labelled ( 2 H, 3 H) peptides through the catalytic asymmetric reduction of Δ Z Trp containing peptides, using rhodium complexes with chiral diphosphine ligands as the catalysts, is described. Ac-Δ Z Trp-( L )-Phe-OMe is used as a model substrate to study this new route. The (Z)-α,β-dehydropeptide is produced by ( L )-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum in a single step reaction. Diastereomeric excesses up to 98 % have been obtained with ( R , R )-dipamp as ligand in the catalyst. Extremely high stereoselectivities for producing the ( L , L )- or ( D , L )-isomer could be achieved using the appropriate chiral ligands. This method has good potential for stereospecific labelling (deuteration or tritiation) of peptides.
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EZ-configurational assignment of N-acetyl-α,β-dehydroTryptophan ethyl ester produced by L-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum
Tetrahedron Letters, 1996Co-Authors: Akli Hammadi, André Ménez, Roger GenetAbstract:Abstract The steric configuration of the α,β-dehydroTryptophanyl moiety produced by incubation of N-acetyl-L-Tryptophan ethyl ester with L-Tryptophan 2′,3′-oxidase was investigated by 1H-NMR spectroscopy. It adopts unambiguously a Z-geometry.
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L-Tryptophan 2′,3′-Oxidase from Chromobacterium violaceum SUBSTRATE SPECIFICITY AND MECHANISTIC IMPLICATIONS
The Journal of biological chemistry, 1995Co-Authors: Roger Genet, Akli Hammadi, Pierre-henri Bénetti, André MénezAbstract:Abstract L-Tryptophan 2′,3′-oxidase, an amino acid α,β-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C and C carbons of various Tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an α-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of Tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic β-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of α,β-dehydroTryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the Tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct α,β-dehydrogenation of the Tryptophan side chain is proposed.
André Ménez - One of the best experts on this subject based on the ideXlab platform.
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Diastereoselective deuteration of (Z)-α,β-dehydroTryptophanyl-containing biological peptides controlled by chiral rhodium catalysts
Tetrahedron Letters, 1998Co-Authors: Akli Hammadi, André Ménez, Gilles Meunier, Roger GenetAbstract:Abstract The regio- and stereoselective labelling ( 2 H, 3 H) of Tryptophanyl containing biological peptides catalysed by chiral rhodium complexes is described. The Δ Z Trp-residue is directly produced in the peptide by a catalytic process, involving the ( S )-Tryptophan 2′,3′-oxydase from Chromobacterium violaceum . Two biological dehydropentapeptides, pGlu-His-Δ Z Trp-Ser-Tyr and Boc-βAla-Δ Z Trp-Met-Asp-Phe-NH 2 , have been thus synthesized. Asymmetric deuteration of these compounds was then investigated in the presence of various rhodium catalysts. High stereocontrol was achieved by suitable choice of the chiral ligands. This strategy offers new possibilities for the asymmetric synthesis of labelled ( 2 H, 3 H) peptides without ultimate deprotection step.
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Asymmetric deuteration of N-acetyl-(Z)-α,β-dehydroTryptophan-(L)-phenylalanine methyl ester produced by (L)-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum. A new route for stereospecific labelling of peptides
Tetrahedron, 1997Co-Authors: Akli Hammadi, André Ménez, Roger GenetAbstract:Abstract A novel approach to the synthesis of labelled ( 2 H, 3 H) peptides through the catalytic asymmetric reduction of Δ Z Trp containing peptides, using rhodium complexes with chiral diphosphine ligands as the catalysts, is described. Ac-Δ Z Trp-( L )-Phe-OMe is used as a model substrate to study this new route. The (Z)-α,β-dehydropeptide is produced by ( L )-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum in a single step reaction. Diastereomeric excesses up to 98 % have been obtained with ( R , R )-dipamp as ligand in the catalyst. Extremely high stereoselectivities for producing the ( L , L )- or ( D , L )-isomer could be achieved using the appropriate chiral ligands. This method has good potential for stereospecific labelling (deuteration or tritiation) of peptides.
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EZ-configurational assignment of N-acetyl-α,β-dehydroTryptophan ethyl ester produced by L-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum
Tetrahedron Letters, 1996Co-Authors: Akli Hammadi, André Ménez, Roger GenetAbstract:Abstract The steric configuration of the α,β-dehydroTryptophanyl moiety produced by incubation of N-acetyl-L-Tryptophan ethyl ester with L-Tryptophan 2′,3′-oxidase was investigated by 1H-NMR spectroscopy. It adopts unambiguously a Z-geometry.
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L-Tryptophan 2′,3′-Oxidase from Chromobacterium violaceum SUBSTRATE SPECIFICITY AND MECHANISTIC IMPLICATIONS
The Journal of biological chemistry, 1995Co-Authors: Roger Genet, Akli Hammadi, Pierre-henri Bénetti, André MénezAbstract:Abstract L-Tryptophan 2′,3′-oxidase, an amino acid α,β-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C and C carbons of various Tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an α-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of Tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic β-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of α,β-dehydroTryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the Tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct α,β-dehydrogenation of the Tryptophan side chain is proposed.
Sandro Ghisla - One of the best experts on this subject based on the ideXlab platform.
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l amino acid oxidase from the malayan pit viper calloselasma rhodostoma comparative sequence analysis and characterization of active and inactive forms of the enzyme
FEBS Journal, 2001Co-Authors: Peter Macheroux, Oliver Seth, Claus Bollschweiler, Margarete Schwarz, Lochun Au, Manfred Kurfurst, Sandro GhislaAbstract:Here we report the cDNA-deduced amino-acid sequence of l-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, d-amino-acid oxidases and Tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, Tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl−, prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.
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L-Amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma Comparative sequence analysis and charaterization of active and inactive forms of the enzyme
European Journal of Biochemistry, 2001Co-Authors: Peter Macheroux, Oliver Seth, Claus Bollschweiler, Margarete Schwarz, Manfred Kurfurst, Sandro GhislaAbstract:Here we report the cDNA-deduced amino-acid sequence of l-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, d-amino-acid oxidases and Tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, Tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl−, prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.
Akli Hammadi - One of the best experts on this subject based on the ideXlab platform.
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Diastereoselective deuteration of (Z)-α,β-dehydroTryptophanyl-containing biological peptides controlled by chiral rhodium catalysts
Tetrahedron Letters, 1998Co-Authors: Akli Hammadi, André Ménez, Gilles Meunier, Roger GenetAbstract:Abstract The regio- and stereoselective labelling ( 2 H, 3 H) of Tryptophanyl containing biological peptides catalysed by chiral rhodium complexes is described. The Δ Z Trp-residue is directly produced in the peptide by a catalytic process, involving the ( S )-Tryptophan 2′,3′-oxydase from Chromobacterium violaceum . Two biological dehydropentapeptides, pGlu-His-Δ Z Trp-Ser-Tyr and Boc-βAla-Δ Z Trp-Met-Asp-Phe-NH 2 , have been thus synthesized. Asymmetric deuteration of these compounds was then investigated in the presence of various rhodium catalysts. High stereocontrol was achieved by suitable choice of the chiral ligands. This strategy offers new possibilities for the asymmetric synthesis of labelled ( 2 H, 3 H) peptides without ultimate deprotection step.
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Asymmetric deuteration of N-acetyl-(Z)-α,β-dehydroTryptophan-(L)-phenylalanine methyl ester produced by (L)-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum. A new route for stereospecific labelling of peptides
Tetrahedron, 1997Co-Authors: Akli Hammadi, André Ménez, Roger GenetAbstract:Abstract A novel approach to the synthesis of labelled ( 2 H, 3 H) peptides through the catalytic asymmetric reduction of Δ Z Trp containing peptides, using rhodium complexes with chiral diphosphine ligands as the catalysts, is described. Ac-Δ Z Trp-( L )-Phe-OMe is used as a model substrate to study this new route. The (Z)-α,β-dehydropeptide is produced by ( L )-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum in a single step reaction. Diastereomeric excesses up to 98 % have been obtained with ( R , R )-dipamp as ligand in the catalyst. Extremely high stereoselectivities for producing the ( L , L )- or ( D , L )-isomer could be achieved using the appropriate chiral ligands. This method has good potential for stereospecific labelling (deuteration or tritiation) of peptides.
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EZ-configurational assignment of N-acetyl-α,β-dehydroTryptophan ethyl ester produced by L-Tryptophan 2′,3′-oxidase from Chromobacterium violaceum
Tetrahedron Letters, 1996Co-Authors: Akli Hammadi, André Ménez, Roger GenetAbstract:Abstract The steric configuration of the α,β-dehydroTryptophanyl moiety produced by incubation of N-acetyl-L-Tryptophan ethyl ester with L-Tryptophan 2′,3′-oxidase was investigated by 1H-NMR spectroscopy. It adopts unambiguously a Z-geometry.
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L-Tryptophan 2′,3′-Oxidase from Chromobacterium violaceum SUBSTRATE SPECIFICITY AND MECHANISTIC IMPLICATIONS
The Journal of biological chemistry, 1995Co-Authors: Roger Genet, Akli Hammadi, Pierre-henri Bénetti, André MénezAbstract:Abstract L-Tryptophan 2′,3′-oxidase, an amino acid α,β-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C and C carbons of various Tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an α-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of Tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic β-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of α,β-dehydroTryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the Tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct α,β-dehydrogenation of the Tryptophan side chain is proposed.
