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Robert S Phillips - One of the best experts on this subject based on the ideXlab platform.
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pressure and temperature effects on formation of aminoacrylate intermediates of Tyrosine Phenol Lyase demonstrate reaction dynamics
ACS Catalysis, 2020Co-Authors: Robert S Phillips, Steven Craig, Andrey Kovalevsky, Oksana Gerlits, Kevin L Weiss, Andreea I Iorgu, Derren J HeyesAbstract:The structures of aminoacrylate intermediates of wild-type, F448A mutant, and perdeuterated Tyrosine Phenol-Lyase (TPL) formed from L-Tyrosine, 3-F-L-Tyrosine, S-ethyl-L-cysteine, and L-serine, with 4-hydroxpyridine bound, were determined by X-ray crystallography. All the aminoacrylate Schiff’s base structures in chain A are identical regardless of the substrate used to form them. The 4-hydroxypyridine is also in an identical location, except for F448A TPL, where it is displaced about 1 A due to the increased size of the active site. In chain B, we have found different complexes depending on the substrate. With wild-type TPL, L-Tyrosine gave no density, 3-F-L-Tyrosine gave a gem-diamine, and L-serine gave a gem-diamine, in chain B. S-Ethyl-L-cysteine formed an aminoacrylate in chain B with both wild-type and F448A TPL, but perdeuterated TPL with S-ethyl-L-cysteine formed a gem-diamine of aminoacrylate. The kinetics of aminoacrylate intermediate formation from L-Tyrosine and S-ethyl-L-cysteine were followe...
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crystal structures of wild type and f448a mutant citrobacter freundii Tyrosine Phenol Lyase complexed with a substrate and inhibitors implications for the reaction mechanism
Biochemistry, 2018Co-Authors: Robert S Phillips, Steven CraigAbstract:Tyrosine Phenol-Lyase (TPL; EC 4.1.99.2) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-Tyrosine to Phenol and ammonium pyruvate. We have shown previously that F448A TPL has kcat and kcat/Km values for l-Tyrosine reduced by ∼104-fold [Phillips, R. S., Vita, A., Spivey, J. B., Rudloff, A. P., Driscoll, M. D., and Hay, S. (2016) ACS Catal. 6, 6770–6779]. We have now obtained crystal structures of F448A TPL and complexes with l-alanine, l-methionine, l-phenylalanine, and 3-F-l-Tyrosine at 2.05–2.27 A and the complex of wild-type TPL with l-phenylalanine at 1.8 A. The small domain of F448A TPL, where Phe-448 is located, is more disordered in chain A than in wild-type TPL. The complexes of F448A TPL with l-alanine and l-phenylalanine are in an open conformation in both chains, while the complex with l-methionine is a 52:48 open:closed equilibrium mixture in chain A. Wild-type TPL with l-alanine is closed in chain A and open in chain B, and the complex with l...
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serine 51 residue of citrobacter freundii Tyrosine Phenol Lyase assists in c α proton abstraction and transfer in the reaction with substrate
Biochimie, 2017Co-Authors: Maria V Barbolina, Vitalia V Kulikova, Marina A Tsvetikova, Natalia V Anufrieva, Svetlana V Revtovich, Tatyana V Demidkina, Paul Gollnick, Robert S Phillips, Nicolai G FaleevAbstract:Abstract In the spatial structure of Tyrosine Phenol-Lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l -Tyrosine and 3-fluoro- l -Tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4′-position of the leaving Phenol group in the reaction with the natural substrate.
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ground state destabilization by phe 448 and phe 449 contributes to Tyrosine Phenol Lyase catalysis
ACS Catalysis, 2016Co-Authors: Robert S Phillips, Andrew Vita, Blaine J Spivey, Alexander P Rudloff, Max D DriscollAbstract:The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme–substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant Tyrosine Phenol-Lyase (TPL) form complexes with 3-fluoro-l-Tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20° out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigate...
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Inhibition of Tyrosine Phenol-Lyase by Tyrosine homologues
Amino Acids, 2016Co-Authors: Quang Do, Giang T. Nguyen, Robert S PhillipsAbstract:We have designed, synthesized, and evaluated Tyrosine homologues and their O -methyl derivatives as potential inhibitors for Tyrosine Phenol Lyase (TPL, E.C. 4.1.99.2). Recently, we reported that homologues of tryptophan are potent inhibitors of tryptophan indole-Lyase (tryptophanase, TIL, E.C. 4.1.99.1), with K _i values in the low µM range (Do et al. Arch Biochem Biophys 560:20–26, 2014 ). As the structure and mechanism for TPL is very similar to that of TIL, we postulated that Tyrosine homologues could also be potent inhibitors of TPL. However, we have found that homoTyrosine, bishomoTyrosine, and their corresponding O -methyl derivatives are competitive inhibitors of TPL, which exhibit K _i values in the range of 0.8–1.5 mM. Thus, these compounds are not potent inhibitors, but instead bind with affinities similar to common amino acids, such as phenylalanine or methionine. Pre-steady-state kinetic data were very similar for all compounds tested and demonstrated the formation of an equilibrating mixture of aldimine and quinonoid intermediates upon binding. Interestingly, we also observed a blue-shift for the absorbance peak of external aldimine complexes of all Tyrosine homologues, suggesting possible strain at the active site due to accommodating the elongated side chains.
Tatyana V Demidkina - One of the best experts on this subject based on the ideXlab platform.
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serine 51 residue of citrobacter freundii Tyrosine Phenol Lyase assists in c α proton abstraction and transfer in the reaction with substrate
Biochimie, 2017Co-Authors: Maria V Barbolina, Vitalia V Kulikova, Marina A Tsvetikova, Natalia V Anufrieva, Svetlana V Revtovich, Tatyana V Demidkina, Paul Gollnick, Robert S Phillips, Nicolai G FaleevAbstract:Abstract In the spatial structure of Tyrosine Phenol-Lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l -Tyrosine and 3-fluoro- l -Tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4′-position of the leaving Phenol group in the reaction with the natural substrate.
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Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent Lyases: the unusual case of Tyrosine Phenol-Lyase
Amino Acids, 2011Co-Authors: Vitalia V. Koulikova, Marina A Tsvetikova, Tatyana V Demidkina, Lyudmila N Zakomirdina, Olga I Gogoleva, Elena A. Morozova, Vsevolod V. Komissarov, Yaroslav V. Tkachev, Vladimir P. Timofeev, Nicolai G FaleevAbstract:A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro -2R- and pro -2S protons of glycine in ^2H_2O under the action of Tyrosine Phenol-Lyase (TPL), tryptophan indole-Lyase (TIL) and methionine γ-Lyase (MGL) was undertaken. The kinetics of exchange was monitored using both ^1H- and ^13C-NMR. In the three compared Lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro -2R proton of glycine with respect to the cofactor pyridoxal 5′-phosphate (PLP) plane. Consequently, according to Dunathan’s postulate with all the three enzymes pro -2R proton should exchange faster than does the pro -2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with l -alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.
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crystallographic snapshots of Tyrosine Phenol Lyase show that substrate strain plays a role in c c bond cleavage
Journal of the American Chemical Society, 2011Co-Authors: Dalibor Milic, Tatyana V Demidkina, Nicolai G Faleev, Robert S Phillips, Dubravka Matkoviccalogovic, Alfred A AntsonAbstract:The key step in the enzymatic reaction catalyzed by Tyrosine Phenol-Lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-Tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-Tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 A resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s Phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captu...
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Crystallographic Snapshots of Tyrosine Phenol-Lyase Show That Substrate Strain Plays a Role in C–C Bond Cleavage
Journal of the American Chemical Society, 2011Co-Authors: Dalibor Milić, Tatyana V Demidkina, Nicolai G Faleev, Robert S Phillips, Dubravka Matković-Čalogović, Alfred A AntsonAbstract:The key step in the enzymatic reaction catalyzed by Tyrosine Phenol-Lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-Tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-Tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 A resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s Phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captu...
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insights into the catalytic mechanism of Tyrosine Phenol Lyase from x ray structures of quinonoid intermediates
Journal of Biological Chemistry, 2008Co-Authors: Dalibor Milic, Tatyana V Demidkina, Nicolai G Faleev, Dubravka Matkoviccalogovic, Alfred A AntsonAbstract:Amino acid transformations catalyzed by a number of pyridoxal 5′-phosphate (PLP)-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii Tyrosine Phenol-Lyase with l-alanine and l-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of Phenol during the β-elimination of l-Tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton.
Nicolai G Faleev - One of the best experts on this subject based on the ideXlab platform.
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serine 51 residue of citrobacter freundii Tyrosine Phenol Lyase assists in c α proton abstraction and transfer in the reaction with substrate
Biochimie, 2017Co-Authors: Maria V Barbolina, Vitalia V Kulikova, Marina A Tsvetikova, Natalia V Anufrieva, Svetlana V Revtovich, Tatyana V Demidkina, Paul Gollnick, Robert S Phillips, Nicolai G FaleevAbstract:Abstract In the spatial structure of Tyrosine Phenol-Lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l -Tyrosine and 3-fluoro- l -Tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4′-position of the leaving Phenol group in the reaction with the natural substrate.
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Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent Lyases: the unusual case of Tyrosine Phenol-Lyase
Amino Acids, 2011Co-Authors: Vitalia V. Koulikova, Marina A Tsvetikova, Tatyana V Demidkina, Lyudmila N Zakomirdina, Olga I Gogoleva, Elena A. Morozova, Vsevolod V. Komissarov, Yaroslav V. Tkachev, Vladimir P. Timofeev, Nicolai G FaleevAbstract:A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro -2R- and pro -2S protons of glycine in ^2H_2O under the action of Tyrosine Phenol-Lyase (TPL), tryptophan indole-Lyase (TIL) and methionine γ-Lyase (MGL) was undertaken. The kinetics of exchange was monitored using both ^1H- and ^13C-NMR. In the three compared Lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro -2R proton of glycine with respect to the cofactor pyridoxal 5′-phosphate (PLP) plane. Consequently, according to Dunathan’s postulate with all the three enzymes pro -2R proton should exchange faster than does the pro -2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with l -alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.
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crystallographic snapshots of Tyrosine Phenol Lyase show that substrate strain plays a role in c c bond cleavage
Journal of the American Chemical Society, 2011Co-Authors: Dalibor Milic, Tatyana V Demidkina, Nicolai G Faleev, Robert S Phillips, Dubravka Matkoviccalogovic, Alfred A AntsonAbstract:The key step in the enzymatic reaction catalyzed by Tyrosine Phenol-Lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-Tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-Tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 A resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s Phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captu...
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Crystallographic Snapshots of Tyrosine Phenol-Lyase Show That Substrate Strain Plays a Role in C–C Bond Cleavage
Journal of the American Chemical Society, 2011Co-Authors: Dalibor Milić, Tatyana V Demidkina, Nicolai G Faleev, Robert S Phillips, Dubravka Matković-Čalogović, Alfred A AntsonAbstract:The key step in the enzymatic reaction catalyzed by Tyrosine Phenol-Lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-Tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-Tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 A resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s Phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captu...
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insights into the catalytic mechanism of Tyrosine Phenol Lyase from x ray structures of quinonoid intermediates
Journal of Biological Chemistry, 2008Co-Authors: Dalibor Milic, Tatyana V Demidkina, Nicolai G Faleev, Dubravka Matkoviccalogovic, Alfred A AntsonAbstract:Amino acid transformations catalyzed by a number of pyridoxal 5′-phosphate (PLP)-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii Tyrosine Phenol-Lyase with l-alanine and l-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of Phenol during the β-elimination of l-Tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton.
Moonhee Sung - One of the best experts on this subject based on the ideXlab platform.
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simultaneous improvement of catalytic activity and thermal stability of Tyrosine Phenol Lyase by directed evolution
FEBS Journal, 2009Co-Authors: Sulim Choi, Seungpyo Hong, Moonhee Sung, Jae J SongAbstract:The Tyrosine Phenol-Lyase from Symbiobacterium toebii was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two-fold improved activity, whereas parallel screening after heat treatment at 65 °C identified three mutants with half-inactivation temperatures improved by up to 5.6 °C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three-fold improved activity and half-inactivation temperatures improved by up to 11.2 °C. Sequence analyses revealed that the stability-improved hits included A13V, E83K and T407A mutations, whereas the activity-improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly–screening process, indicating the critical nature of the N-terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer–dimer interface, including the N-terminus, whereas the activity-improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co-improvement of the stability and catalytic activity of the enzyme.
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development of bioreactor system for l Tyrosine synthesis using thermostable Tyrosine Phenol Lyase
Journal of Microbiology and Biotechnology, 2007Co-Authors: Sulim Choi, Seungpyo Hong, Jae Jun Song, Moonhee SungAbstract:: An efficient enzyme system for the synthesis of L-Tyrosine was developed using a fed-batch reactor with continuous feeding of Phenol, pyruvate, and ammonia. A thermo- and chemostable Tyrosine Phenol-Lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM Phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M Phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-Tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-Tyrosine was 94% on the basis of the supplied Phenol.
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inactivation of Tyrosine Phenol Lyase by pictet spengler reaction and alleviation by t15a mutation on intertwined n terminal arm
FEBS Journal, 2006Co-Authors: Seungpyo Hong, Jae Jun Song, Hyeon Su Ro, Moonhee SungAbstract:Citrobacter freundiil-Tyrosine Phenol-Lyase (TPL) was inactivated by a Pictet–Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40 °C, respectively, which was 15 °C lower than when using l-Tyrosine as the substrate. The half-lives at 45 °C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mm l-DOPA. On treatment with excess pyridoxal-5′-phosphate (PLP), the l-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet–Spengler condensation with l-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (Km,PLP) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 °C, implying that the loss of the cofactor with the Pictet–Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.
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Inactivation of Tyrosine Phenol‐Lyase by Pictet–Spengler reaction and alleviation by T15A mutation on intertwined N‐terminal arm
FEBS Journal, 2006Co-Authors: Seungpyo Hong, Jae Jun Song, Hyeon Su Ro, Moonhee SungAbstract:Citrobacter freundiil-Tyrosine Phenol-Lyase (TPL) was inactivated by a Pictet–Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40 °C, respectively, which was 15 °C lower than when using l-Tyrosine as the substrate. The half-lives at 45 °C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mm l-DOPA. On treatment with excess pyridoxal-5′-phosphate (PLP), the l-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet–Spengler condensation with l-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (Km,PLP) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 °C, implying that the loss of the cofactor with the Pictet–Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.
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enhanced stability of Tyrosine Phenol Lyase from symbiobacterium toebii by dna shuffling
Journal of Microbiology and Biotechnology, 2004Co-Authors: Jae Jun Song, Moonhee SungAbstract:Tyrosine Phenol-Lyase (TPL) is a useful enzyme for the synthesis of pharmaceutical aromatic amino acids. In the current study, sequential DNA shuffling and screening were used to enhance the stability of TPL. Twenty-thousand mutants were screened, and several improved variants were isolated. One variant named A13V, in which the 13 th amino acid alanine was substituted by valine, exhibited a higher temperature and denaturant stability than the wild-type TPL. The purified mutant TPL, A13V, retained about 60% of its activity at 76°C, whereas the activity of the wild-type TPL decreased to less than 20% at the same temperature. Plus, A13V exhibited about 50% activity with 3 M urea, while the wild-type TPL lost almost all its catalytic activity, indicating an increased denaturant tolerance in the mutant A13V. It is speculated that the substitution of Val for the Ala in the β-strand of the N-terminal arm was responsible for the heightened stabilization, and that the current results will contribute to further research on the structural stability of TPL.
Seungpyo Hong - One of the best experts on this subject based on the ideXlab platform.
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simultaneous improvement of catalytic activity and thermal stability of Tyrosine Phenol Lyase by directed evolution
FEBS Journal, 2009Co-Authors: Sulim Choi, Seungpyo Hong, Moonhee Sung, Jae J SongAbstract:The Tyrosine Phenol-Lyase from Symbiobacterium toebii was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two-fold improved activity, whereas parallel screening after heat treatment at 65 °C identified three mutants with half-inactivation temperatures improved by up to 5.6 °C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three-fold improved activity and half-inactivation temperatures improved by up to 11.2 °C. Sequence analyses revealed that the stability-improved hits included A13V, E83K and T407A mutations, whereas the activity-improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly–screening process, indicating the critical nature of the N-terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer–dimer interface, including the N-terminus, whereas the activity-improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co-improvement of the stability and catalytic activity of the enzyme.
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development of bioreactor system for l Tyrosine synthesis using thermostable Tyrosine Phenol Lyase
Journal of Microbiology and Biotechnology, 2007Co-Authors: Sulim Choi, Seungpyo Hong, Jae Jun Song, Moonhee SungAbstract:: An efficient enzyme system for the synthesis of L-Tyrosine was developed using a fed-batch reactor with continuous feeding of Phenol, pyruvate, and ammonia. A thermo- and chemostable Tyrosine Phenol-Lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM Phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M Phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-Tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-Tyrosine was 94% on the basis of the supplied Phenol.
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inactivation of Tyrosine Phenol Lyase by pictet spengler reaction and alleviation by t15a mutation on intertwined n terminal arm
FEBS Journal, 2006Co-Authors: Seungpyo Hong, Jae Jun Song, Hyeon Su Ro, Moonhee SungAbstract:Citrobacter freundiil-Tyrosine Phenol-Lyase (TPL) was inactivated by a Pictet–Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40 °C, respectively, which was 15 °C lower than when using l-Tyrosine as the substrate. The half-lives at 45 °C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mm l-DOPA. On treatment with excess pyridoxal-5′-phosphate (PLP), the l-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet–Spengler condensation with l-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (Km,PLP) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 °C, implying that the loss of the cofactor with the Pictet–Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.
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Inactivation of Tyrosine Phenol‐Lyase by Pictet–Spengler reaction and alleviation by T15A mutation on intertwined N‐terminal arm
FEBS Journal, 2006Co-Authors: Seungpyo Hong, Jae Jun Song, Hyeon Su Ro, Moonhee SungAbstract:Citrobacter freundiil-Tyrosine Phenol-Lyase (TPL) was inactivated by a Pictet–Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40 °C, respectively, which was 15 °C lower than when using l-Tyrosine as the substrate. The half-lives at 45 °C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mm l-DOPA. On treatment with excess pyridoxal-5′-phosphate (PLP), the l-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet–Spengler condensation with l-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (Km,PLP) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 °C, implying that the loss of the cofactor with the Pictet–Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.
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characterization of thermostable Tyrosine Phenol Lyase from an obligatory symbiotic thermophile symbiobacterium sp sc 1
Journal of Biochemistry and Molecular Biology, 1999Co-Authors: Seungpyo Hong, Mi Sun Kwak, Nobuyoshi Esaki, Moonhee SungAbstract:Tyrosine Phenol-Lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, , for PLP was determined with the apoenzyme to be about . The isoelectric point was 4.9. The optimal temperature and pH for the -elimination of L-Tyrosine were found to be and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-Tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, -chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, -elimination, was the only D-amino acid racemized by the enzyme. The values for L-Tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, -chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.
