The Experts below are selected from a list of 1866 Experts worldwide ranked by ideXlab platform
Mengsu Yang - One of the best experts on this subject based on the ideXlab platform.
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surface plasmon resonance measurement of pyridoxal kinase Pyridoxamine binding on self assembled monolayer
Sensors and Actuators B-chemical, 2002Co-Authors: Hellas C M Yau, Mengsu YangAbstract:Abstract Pyridoxamine-modified surface was prepared from self-assembly of Pyridoxamine-linked 11-mercapto-undecan-1-one (PMU), which is synthesized via the chemistry of EDC/NHS activating 11-mercapto-undecanoic acid to link with Pyridoxamine, on gold surface for the study of interaction between Pyridoxamine and pyridoxal kinase (PK). This interaction was monitored by a low-cost red light-emitting-diode-based surface plasmon resonance technique. Addition of either 11-mercapto-undecan-1-ol (MU) or 1-dodecanethiol (DDT) to PMU for generating a mixed film can improve the performance of the monolayer of PMU for PK binding. Mixture of PMU and DDT in the mole ratio as low as 1:5 can achieve a significant enhancement (at least 89%) to the responses of PK binding.
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study of substrate enzyme interaction between immobilized Pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor
Biochimica et Biophysica Acta, 2002Co-Authors: Chi-chun Fong, Y. C. Leung, Man Sau Wong, Wanping Lai, Mengsu YangAbstract:Abstract Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B6. Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, Pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized Pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na+, K+) and divalent cations (Mn2+, Zn2+, Mg2+) on the binding kinetics were determined. Optimal pH for PK–Pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K+ increased and Na+ decreased the binding affinity (KA) of PK to immobilized Pyridoxamine, all divalent cations increased the KA of PK for Pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B6 analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>Pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.
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Characterization of Recombinant Porcine Pyridoxal Kinase using Surface Plasmon Resonance Biosensor Technique
Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000Co-Authors: Chi-chun Fong, W. P. Lai, Mengsu Yang, Y. C. Leung, Man Sau WongAbstract:In the present study, surface plasmon resonance biosensor was employed to characterize the binding interaction between wild type porcine Pyridoxal Kinase (PK) and an immobilized substrate, Pyridoxamine. The binding of PK to the immobilized Pyridoxamine was followed in real time and the kinetic parameters were derived from the sensorgram using the BlAevaluationTM software. At pH 7.4, the association rate constant and dissociation rate constant of the wild-type PK for immobilized Pyridoxamine are 7.25 x 103±0.35 x 103M-1s-1 and 5.01 x 10-3±0.93 x 10-3s-1respectively. The equilibrium affinity constant of the wild-type PK is 1.45 x 106M-1. The effect of buffer pH on the binding kinetic parameters was also determined.
Paul A. Voziyan - One of the best experts on this subject based on the ideXlab platform.
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Pyridoxamine protects protein backbone from oxidative fragmentation
Biochemical and Biophysical Research Communications, 2011Co-Authors: Sergei V. Chetyrkin, Billy G. Hudson, Missy Mathis, Hayes W Mcdonald, Xavier Shackelford, Paul A. VoziyanAbstract:Oxidative damage to proteins is one of the major pathogenic mechanisms in many chronic diseases. Therefore, inhibition of this oxidative damage can be an important part of therapeutic strategies. Pyridoxamine (PM), a prospective drug for treatment of diabetic nephropathy, has been previously shown to inhibit several oxidative and glycoxidative pathways, thus protecting amino acid side chains of the proteins from oxidative damage. Here, we demonstrated that PM can also protect protein backbone from fragmentation induced via different oxidative mechanisms including autoxidation of glucose. This protection was due to hydroxyl radical scavenging by PM and may contribute to PM therapeutic effects shown in clinical trials.
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Pyridoxamine protects intestinal epithelium from ionizing radiation induced apoptosis
Free Radical Biology and Medicine, 2009Co-Authors: D Thotala, Paul A. Voziyan, Sergei V. Chetyrkin, Billy G. Hudson, Dennis E Hallahan, Eugenia M YazlovitskayaAbstract:Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are the major causes of biological tissue damage during exposure to ionizing radiation (IR). The existing strategies to protect normal tissues from the detrimental effects of IR suffer from several shortcomings including highly toxic side effects, unfavorable administration routes, and low efficacy. These shortcomings emphasize a need for radioprotective treatments that combine effectiveness with safety and ease of use. In this paper, we demonstrate that Pyridoxamine, a ROS and RCS scavenger with a very favorable safety profile, can inhibit IR-induced gastrointestinal epithelial apoptosis in cell culture and in an animal model. Pyridoxamine was more effective at protecting from radiation-induced apoptosis than amifostine, a synthetic thiol compound and the only FDA-approved radioprotector. We suggest that Pyridoxamine has potential as an effective and safe radioprotective agent.
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Pyridoxamine protects proteins from functional damage by 3 deoxyglucosone mechanism of action of Pyridoxamine
Biochemistry, 2008Co-Authors: Sergei V. Chetyrkin, Billy G. Hudson, Wenhui Zhang, And Anthony S Serianni, Paul A. VoziyanAbstract:Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell−matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952−2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyg...
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Pyridoxamine protects proteins from functional damage by 3-deoxyglucosone: mechanism of action of Pyridoxamine.
Biochemistry, 2007Co-Authors: Sergei V. Chetyrkin, Billy G. Hudson, Wenhui Zhang, And Anthony S Serianni, Paul A. VoziyanAbstract:Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell-matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952-2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyglucosone (3-DG) can damage protein functionality, including the ability of collagen IV to interact with glomerular mesangial cells. We have also demonstrated that PM can protect against 3-DG-induced protein damage via a novel mechanism that includes transient adduction of 3-DG by PM followed by irreversible PM-mediated oxidative cleavage of 3-DG. Our results suggest that, in diabetic nephropathy, the therapeutic effect of PM is achieved, in part, via protection of renal cell-matrix interactions from damage by a variety of RCS. Our data emphasize the potential importance of the contribution by 3-DG, along with other more reactive RCS, to this pathogenic mechanism.
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Pyridoxamine analogues scavenge lipid derived γ ketoaldehydes and protect against h2o2 mediated cytotoxicity
Biochemistry, 2006Co-Authors: Sean S. Davies, Olivier Boutaud, John A. Oates, Irene Zagolikapitte, Paul A. Voziyan, Billy G. Hudson, Venkataraman Amarnath, Eric J Brantley, Jackson L RobertsAbstract:Isoketals and levuglandins are highly reactive γ-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether Pyridoxamine, which has been shown to scavenge α-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive γ-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic γ-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of Pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogs of Pyridoxamine, salicylamine and 5’O-pentylPyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of Pyridoxamine or its analogs, Pyridoxamine-isoketal adducts were found in significantly greater abundance than the Pyridoxamine-N-acyl adducts formed by α-ketoaldehyde scavenging. Therefore, Pyridoxamine and its analogs appear to preferentially scavenge γ-ketoaldehydes. Both Pyridoxamine and its lipophilic analogs inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic Pyridoxamine analogs provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of Pyridoxamine and lipophilic Pyridoxamine analogs to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.
Ronald Breslow - One of the best experts on this subject based on the ideXlab platform.
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dendrimers in solution can have their remote catalytic groups folded back into the core enantioselective transaminations by dendritic enzyme mimics ii
Bioorganic & Medicinal Chemistry Letters, 2009Co-Authors: Sujun Wei, Jianing Wang, Scott Venhuizen, Rachid Skouta, Ronald BreslowAbstract:PAMAM dendrimers with double thioether arms have been synthesized with a Pyridoxamine core and terminal chiral amino groups. Transamination to afford natural isomers of phenylalanine and alanine induced enantioselectivity by the peripheral chiral caps, supporting a computer model that indicates folding of dendrimer chains back into the core.
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transamination reactions with multiple turnovers catalyzed by hydrophobic Pyridoxamine cofactors in the presence of polyethylenimine polymers
Journal of the American Chemical Society, 2004Co-Authors: Lei Liu, Wenjun Zhou, Jason J Chruma, Ronald BreslowAbstract:Pyridoxamines carrying hydrophobic side chains reversibly bind into the hydrophobic core of polyethylenimines and transaminate ketoacids to amino acids with as much as a 725000-fold rate acceleration. Turnover catalysis was achieved by sacrificial oxidative decarboxylation of C-substituted amino acids, which reconverted the pyridoxals to Pyridoxamines.
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Dendrimeric Pyridoxamine enzyme mimics.
Journal of the American Chemical Society, 2003Co-Authors: Ronald BreslowAbstract:PAMAM dendrimers from generations 1−6 were synthesized with Pyridoxamine in their core. They transaminated pyruvic and phenylpyruvic acids in water to alanine and phenylalanine, respectively, with Michaelis−Menten kinetics and high effectiveness compared with simple Pyridoxamine. The largest dendrimerssimilar in size to some globular proteinswere comparable in effectiveness to a previous polyethylenimine (PEI)−Pyridoxamine catalyst, and to a protein−Pyridoxamine catalyst, but not as effective as a previous PEI−Pyridoxamine carrying lauryl hydrophobic groups. The new catalysts showed both general acid/base catalysis by their amino groups and hydrophobic binding of the phenylpyruvate substrate.
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a potent polymer Pyridoxamine enzyme mimic
Journal of the American Chemical Society, 2002Co-Authors: Ronald BreslowAbstract:An enzyme mimic consisting of Pyridoxamines covalently linked to polyethyleneimine carrying long-chain alkyl groups converts pyruvic acid to dl-alanine with as much as an 8000-fold acceleration relative to the reaction with simple Pyridoxamine at the same Pyridoxamine concentration. The acceleration by polymer is a strong function of the length of the alkyl chains that are appended. The polymer furnishes acid and base groups to catalyze the proton transfers that are involved in transamination.
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reversal of optical induction in transamination by regioisomeric bifunctionalized cyclodextrins
Bioorganic & Medicinal Chemistry, 1999Co-Authors: Elisabetta Fasella, Steven D Dong, Ronald BreslowAbstract:Abstract Two isomeric compounds have been synthesized carrying a Pyridoxamine on C-6 of β-cyclodextrin and an imidazole unit on C-6 of the neighboring glucose residue. Each one stereoselectively transaminates phenylpyruvic acid to produce phenylalanine, and with opposite stereochemical preferences. Structure determinations by X-ray crystallography and NMR spectroscopy indicate that the imidazole units serve to block proton addition from their side, rather than acting to protonate the transamination intermediates. Related cyclodextrin–Pyridoxamine compounds had been reported carrying ethylenediamine units instead of imidazoles, and high enantioselectivities in transamination were claimed. Our work indicates that these claims are incorrect, and that only poor selectivities are seen that are often unrelated to the position of the ethylenediamine units. Neither of these transaminating systems yet approaches the enantioselectivity seen with a Pyridoxamine carrying a chirally mounted internal base group.
Billy G. Hudson - One of the best experts on this subject based on the ideXlab platform.
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Pyridoxamine protects protein backbone from oxidative fragmentation
Biochemical and Biophysical Research Communications, 2011Co-Authors: Sergei V. Chetyrkin, Billy G. Hudson, Missy Mathis, Hayes W Mcdonald, Xavier Shackelford, Paul A. VoziyanAbstract:Oxidative damage to proteins is one of the major pathogenic mechanisms in many chronic diseases. Therefore, inhibition of this oxidative damage can be an important part of therapeutic strategies. Pyridoxamine (PM), a prospective drug for treatment of diabetic nephropathy, has been previously shown to inhibit several oxidative and glycoxidative pathways, thus protecting amino acid side chains of the proteins from oxidative damage. Here, we demonstrated that PM can also protect protein backbone from fragmentation induced via different oxidative mechanisms including autoxidation of glucose. This protection was due to hydroxyl radical scavenging by PM and may contribute to PM therapeutic effects shown in clinical trials.
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Pyridoxamine protects intestinal epithelium from ionizing radiation induced apoptosis
Free Radical Biology and Medicine, 2009Co-Authors: D Thotala, Paul A. Voziyan, Sergei V. Chetyrkin, Billy G. Hudson, Dennis E Hallahan, Eugenia M YazlovitskayaAbstract:Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are the major causes of biological tissue damage during exposure to ionizing radiation (IR). The existing strategies to protect normal tissues from the detrimental effects of IR suffer from several shortcomings including highly toxic side effects, unfavorable administration routes, and low efficacy. These shortcomings emphasize a need for radioprotective treatments that combine effectiveness with safety and ease of use. In this paper, we demonstrate that Pyridoxamine, a ROS and RCS scavenger with a very favorable safety profile, can inhibit IR-induced gastrointestinal epithelial apoptosis in cell culture and in an animal model. Pyridoxamine was more effective at protecting from radiation-induced apoptosis than amifostine, a synthetic thiol compound and the only FDA-approved radioprotector. We suggest that Pyridoxamine has potential as an effective and safe radioprotective agent.
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Pyridoxamine protects proteins from functional damage by 3 deoxyglucosone mechanism of action of Pyridoxamine
Biochemistry, 2008Co-Authors: Sergei V. Chetyrkin, Billy G. Hudson, Wenhui Zhang, And Anthony S Serianni, Paul A. VoziyanAbstract:Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell−matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952−2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyg...
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Pyridoxamine protects proteins from functional damage by 3-deoxyglucosone: mechanism of action of Pyridoxamine.
Biochemistry, 2007Co-Authors: Sergei V. Chetyrkin, Billy G. Hudson, Wenhui Zhang, And Anthony S Serianni, Paul A. VoziyanAbstract:Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell-matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952-2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyglucosone (3-DG) can damage protein functionality, including the ability of collagen IV to interact with glomerular mesangial cells. We have also demonstrated that PM can protect against 3-DG-induced protein damage via a novel mechanism that includes transient adduction of 3-DG by PM followed by irreversible PM-mediated oxidative cleavage of 3-DG. Our results suggest that, in diabetic nephropathy, the therapeutic effect of PM is achieved, in part, via protection of renal cell-matrix interactions from damage by a variety of RCS. Our data emphasize the potential importance of the contribution by 3-DG, along with other more reactive RCS, to this pathogenic mechanism.
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Pyridoxamine analogues scavenge lipid derived γ ketoaldehydes and protect against h2o2 mediated cytotoxicity
Biochemistry, 2006Co-Authors: Sean S. Davies, Olivier Boutaud, John A. Oates, Irene Zagolikapitte, Paul A. Voziyan, Billy G. Hudson, Venkataraman Amarnath, Eric J Brantley, Jackson L RobertsAbstract:Isoketals and levuglandins are highly reactive γ-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether Pyridoxamine, which has been shown to scavenge α-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive γ-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic γ-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of Pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogs of Pyridoxamine, salicylamine and 5’O-pentylPyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of Pyridoxamine or its analogs, Pyridoxamine-isoketal adducts were found in significantly greater abundance than the Pyridoxamine-N-acyl adducts formed by α-ketoaldehyde scavenging. Therefore, Pyridoxamine and its analogs appear to preferentially scavenge γ-ketoaldehydes. Both Pyridoxamine and its lipophilic analogs inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic Pyridoxamine analogs provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of Pyridoxamine and lipophilic Pyridoxamine analogs to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.
Toshiharu Yagi - One of the best experts on this subject based on the ideXlab platform.
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engineering mesorhizobium loti Pyridoxamine pyruvate aminotransferase for production of Pyridoxamine with l glutamate as an amino donor
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Yu Yoshikane, Nana Yokochi, Asuka Tamura, Khalil Ellouze, Eitora Yamamura, Hanae Mizunaga, Noboru Fujimoto, Keiji Sakamoto, Yoshihiro Sawa, Toshiharu YagiAbstract:Abstract Pyridoxamine–pyruvate aminotransferase (PPAT), a novel pyridoxal 5′-phosphate-independent aminotransferase, reversibly catalyzes the transfer of an amino group between Pyridoxamine and pyruvate to generate pyridoxal and l -alanine. The enzyme can be used for synthesis of Pyridoxamine, a promising candidate for prophylaxis and treatment of diabetic complications. A disadvantage of PPAT for industrial application to the synthesis is that it requires an expensive amino acid l -alanine as an amino donor. Here, mutated PPATs with a high activity toward 2-oxoglutarate (and hence toward l -glutamate) were prepared by a rational design plus random mutagenesis of the wild-type PPAT because l -glutamate is readily and economically available. The PPAT(Y35H/V70R/F247C) showed 9.1-fold lower Km and 4.3-fold higher kcat values than those of the wild-type PPAT. The model of the complex of mutated PPAT and pyridoxyl- l -glutamate showed that γ-carboxyl group of l -glutamate was hydrogen-bound with an imidazole group of His35. The production of Pyridoxamine from pyridoxal with transformed Escherichia coli cells expressing the mutated PPAT did not correlate with the kcat value or catalytic efficiency of the mutated PPAT but with Km value at a low level. E. coli cells expressing the PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of pyridoxal into Pyridoxamine at 30 °C with l -glutamate as an amino donor.
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crystal structure of Pyridoxamine pyruvate aminotransferase from mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between Pyridoxamine and pyruvate to form pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with Pyridoxamine, pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5′-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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Crystal structure of Pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099
The Journal of biological chemistry, 2007Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between Pyridoxamine and pyruvate to form pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with Pyridoxamine, pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5'-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
