The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Robert Huber - One of the best experts on this subject based on the ideXlab platform.
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the ligand induced structural changes of human l arginine glycine amidinotransferase a mutational and crystallographic study
Journal of Biological Chemistry, 1999Co-Authors: Erich Fritsche, Andreas Humm, Robert HuberAbstract:Human L-arginine:glycine amidinotransferase (AT) shows large structural changes of the 300-flap and of helix H9 upon binding of L-arginine and L-ornithine, described as a closed and an open conformation (Humm, A., Fritsche, E., Steinbacher, S., and Huber, R. (1997) EMBO J. 16, 3373-3385). To elucidate the structural basis of these induced-fit movements, the x-ray structures of AT in complex with the amidino acceptor glycine and its analogs gamma-aminobutyric acid and delta-aminovaleric acid, as well as in complex with the amidino donor analogs L-alanine, L-alpha-aminobutyric acid, and L-norvaline, have been solved at 2.6-, 2.5-, 2.37-, 2.3-, 2.5-, and 2.4-A resolutions, respectively. The latter three compounds were found to stabilize the open conformer. The glycine analogs bind in a distinct manner and do not induce the transition to the open state. The complex with glycine revealed a third binding mode, reflecting the rather broad substrate specificity of AT. These findings identified a role for the alpha-amino group of the ligand in stabilizing the open conformer. The kinetic, structural, and thermodynamic properties of the mutants ATDeltaM302 and ATDelta11 (lacks 11 residues of H9) confirmed the key role of Asn300 and suggest that in mammalian Amidinotransferases, the role of helix H9 is in accelerating amidino transfer by an induced-fit mechanism. Helix H9 does not add to the stability of the protein.
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Crystal Structure of l-Arginine:Inosamine-Phosphate Amidinotransferase StrB1 from Streptomyces griseus: An Enzyme Involved in Streptomycin Biosynthesis‡
Biochemistry, 1998Co-Authors: Erich Fritsche, Andreas Humm, Andreas Bergner, Wolfgang Piepersberg, Robert HuberAbstract:Inosamine-phosphate Amidinotransferases catalyze two nonconsecutive transamidination reactions in the biosynthesis of the streptomycin family of antibiotics. l-Arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus (StrB1) was cloned as an N-terminal hexa-histidine fusion protein, purified by affinity chromatography, and crystallized, and its crystal structure was solved by Patterson search methods at 3.1 A resolution. The structure is composed of five ββαβ-modules which are arranged circularly into a pseudo-5-fold symmetric particle. The three-dimensional structure is closely related to the structure of human l-arginine:glycine amidinotransferase (AT), but five loops (the 40-, 170-, 220-, 250-, and 270-loop) are organized very differently. The major changes are found in loops around the active site which open the narrow active site channel of AT to form an open and solvent-exposed cavity. In particular, module II of StrB1 is AT-like but lacks a 10-residue α-helix in the 170-loop....
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Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
European journal of biochemistry, 1997Co-Authors: Erich Fritsche, Andreas Humm, Robert HuberAbstract:L-Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active-site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recom-binant human L-arginine : glycine amidinotransferase were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28-nm, 0.29-nm and 0.236-nm resolution, respectively. The mutation of active-site residues which are involved in substrate-binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000-fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild-type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition-state stabilization, and not due to major conformational rearrangements of the protein. These results show that a His-Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain-like cysteine proteinases.
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CRYSTAL STRUCTURE AND MECHANISM OF HUMAN L-ARGININE: GLYCINE AMIDINOTRANSFERASE: A MITOCHONDRIAL ENZYME INVOLVED IN CREATINE BIOSYNTHESIS
The EMBO journal, 1997Co-Authors: Andreas Humm, Erich Fritsche, Stefan Steinbacher, Robert HuberAbstract:L-arginine:glycine amidinotransferase (AT) catalyses the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the immediate precursor of creatine. We have determined the crystal structure of the recombinant human enzyme by multiple isomorphous replacement at 1.9 A resolution. A telluromethionine derivative was used in sequence assignment. The structure of AT reveals a new fold with 5-fold pseudosymmetry of circularly arranged betabeta alphabeta-modules. These enclose the active site compartment, which is accessible only through a narrow channel. The overall structure resembles a basket with handles that are formed from insertions into the betabeta alphabeta-modules. Binding of L-ornithine, a product inhibitor, reveals a marked induced-fit mechanism, with a loop at the active site entrance changing its conformation accompanied by a shift of an alpha-helix by -4 A. Binding of the arginine educt to the inactive mutant C407A shows a similar mode of binding. A reaction mechanism with a catalytic triad Cys-His-Asp is proposed on the basis of substrate and product bound states.
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recombinant expression and isolation of human l arginine glycine amidinotransferase and identification of its active site cysteine residue
Biochemical Journal, 1997Co-Authors: Andreas Humm, Erich Fritsche, Karlheinz Mann, Martin Gohl, Robert HuberAbstract:Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine:glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coli with two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.
Andreas Humm - One of the best experts on this subject based on the ideXlab platform.
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the ligand induced structural changes of human l arginine glycine amidinotransferase a mutational and crystallographic study
Journal of Biological Chemistry, 1999Co-Authors: Erich Fritsche, Andreas Humm, Robert HuberAbstract:Human L-arginine:glycine amidinotransferase (AT) shows large structural changes of the 300-flap and of helix H9 upon binding of L-arginine and L-ornithine, described as a closed and an open conformation (Humm, A., Fritsche, E., Steinbacher, S., and Huber, R. (1997) EMBO J. 16, 3373-3385). To elucidate the structural basis of these induced-fit movements, the x-ray structures of AT in complex with the amidino acceptor glycine and its analogs gamma-aminobutyric acid and delta-aminovaleric acid, as well as in complex with the amidino donor analogs L-alanine, L-alpha-aminobutyric acid, and L-norvaline, have been solved at 2.6-, 2.5-, 2.37-, 2.3-, 2.5-, and 2.4-A resolutions, respectively. The latter three compounds were found to stabilize the open conformer. The glycine analogs bind in a distinct manner and do not induce the transition to the open state. The complex with glycine revealed a third binding mode, reflecting the rather broad substrate specificity of AT. These findings identified a role for the alpha-amino group of the ligand in stabilizing the open conformer. The kinetic, structural, and thermodynamic properties of the mutants ATDeltaM302 and ATDelta11 (lacks 11 residues of H9) confirmed the key role of Asn300 and suggest that in mammalian Amidinotransferases, the role of helix H9 is in accelerating amidino transfer by an induced-fit mechanism. Helix H9 does not add to the stability of the protein.
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Crystal Structure of l-Arginine:Inosamine-Phosphate Amidinotransferase StrB1 from Streptomyces griseus: An Enzyme Involved in Streptomycin Biosynthesis‡
Biochemistry, 1998Co-Authors: Erich Fritsche, Andreas Humm, Andreas Bergner, Wolfgang Piepersberg, Robert HuberAbstract:Inosamine-phosphate Amidinotransferases catalyze two nonconsecutive transamidination reactions in the biosynthesis of the streptomycin family of antibiotics. l-Arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus (StrB1) was cloned as an N-terminal hexa-histidine fusion protein, purified by affinity chromatography, and crystallized, and its crystal structure was solved by Patterson search methods at 3.1 A resolution. The structure is composed of five ββαβ-modules which are arranged circularly into a pseudo-5-fold symmetric particle. The three-dimensional structure is closely related to the structure of human l-arginine:glycine amidinotransferase (AT), but five loops (the 40-, 170-, 220-, 250-, and 270-loop) are organized very differently. The major changes are found in loops around the active site which open the narrow active site channel of AT to form an open and solvent-exposed cavity. In particular, module II of StrB1 is AT-like but lacks a 10-residue α-helix in the 170-loop....
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Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
European journal of biochemistry, 1997Co-Authors: Erich Fritsche, Andreas Humm, Robert HuberAbstract:L-Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active-site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recom-binant human L-arginine : glycine amidinotransferase were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28-nm, 0.29-nm and 0.236-nm resolution, respectively. The mutation of active-site residues which are involved in substrate-binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000-fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild-type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition-state stabilization, and not due to major conformational rearrangements of the protein. These results show that a His-Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain-like cysteine proteinases.
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CRYSTAL STRUCTURE AND MECHANISM OF HUMAN L-ARGININE: GLYCINE AMIDINOTRANSFERASE: A MITOCHONDRIAL ENZYME INVOLVED IN CREATINE BIOSYNTHESIS
The EMBO journal, 1997Co-Authors: Andreas Humm, Erich Fritsche, Stefan Steinbacher, Robert HuberAbstract:L-arginine:glycine amidinotransferase (AT) catalyses the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the immediate precursor of creatine. We have determined the crystal structure of the recombinant human enzyme by multiple isomorphous replacement at 1.9 A resolution. A telluromethionine derivative was used in sequence assignment. The structure of AT reveals a new fold with 5-fold pseudosymmetry of circularly arranged betabeta alphabeta-modules. These enclose the active site compartment, which is accessible only through a narrow channel. The overall structure resembles a basket with handles that are formed from insertions into the betabeta alphabeta-modules. Binding of L-ornithine, a product inhibitor, reveals a marked induced-fit mechanism, with a loop at the active site entrance changing its conformation accompanied by a shift of an alpha-helix by -4 A. Binding of the arginine educt to the inactive mutant C407A shows a similar mode of binding. A reaction mechanism with a catalytic triad Cys-His-Asp is proposed on the basis of substrate and product bound states.
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recombinant expression and isolation of human l arginine glycine amidinotransferase and identification of its active site cysteine residue
Biochemical Journal, 1997Co-Authors: Andreas Humm, Erich Fritsche, Karlheinz Mann, Martin Gohl, Robert HuberAbstract:Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine:glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coli with two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.
Erich Fritsche - One of the best experts on this subject based on the ideXlab platform.
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the ligand induced structural changes of human l arginine glycine amidinotransferase a mutational and crystallographic study
Journal of Biological Chemistry, 1999Co-Authors: Erich Fritsche, Andreas Humm, Robert HuberAbstract:Human L-arginine:glycine amidinotransferase (AT) shows large structural changes of the 300-flap and of helix H9 upon binding of L-arginine and L-ornithine, described as a closed and an open conformation (Humm, A., Fritsche, E., Steinbacher, S., and Huber, R. (1997) EMBO J. 16, 3373-3385). To elucidate the structural basis of these induced-fit movements, the x-ray structures of AT in complex with the amidino acceptor glycine and its analogs gamma-aminobutyric acid and delta-aminovaleric acid, as well as in complex with the amidino donor analogs L-alanine, L-alpha-aminobutyric acid, and L-norvaline, have been solved at 2.6-, 2.5-, 2.37-, 2.3-, 2.5-, and 2.4-A resolutions, respectively. The latter three compounds were found to stabilize the open conformer. The glycine analogs bind in a distinct manner and do not induce the transition to the open state. The complex with glycine revealed a third binding mode, reflecting the rather broad substrate specificity of AT. These findings identified a role for the alpha-amino group of the ligand in stabilizing the open conformer. The kinetic, structural, and thermodynamic properties of the mutants ATDeltaM302 and ATDelta11 (lacks 11 residues of H9) confirmed the key role of Asn300 and suggest that in mammalian Amidinotransferases, the role of helix H9 is in accelerating amidino transfer by an induced-fit mechanism. Helix H9 does not add to the stability of the protein.
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Crystal Structure of l-Arginine:Inosamine-Phosphate Amidinotransferase StrB1 from Streptomyces griseus: An Enzyme Involved in Streptomycin Biosynthesis‡
Biochemistry, 1998Co-Authors: Erich Fritsche, Andreas Humm, Andreas Bergner, Wolfgang Piepersberg, Robert HuberAbstract:Inosamine-phosphate Amidinotransferases catalyze two nonconsecutive transamidination reactions in the biosynthesis of the streptomycin family of antibiotics. l-Arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus (StrB1) was cloned as an N-terminal hexa-histidine fusion protein, purified by affinity chromatography, and crystallized, and its crystal structure was solved by Patterson search methods at 3.1 A resolution. The structure is composed of five ββαβ-modules which are arranged circularly into a pseudo-5-fold symmetric particle. The three-dimensional structure is closely related to the structure of human l-arginine:glycine amidinotransferase (AT), but five loops (the 40-, 170-, 220-, 250-, and 270-loop) are organized very differently. The major changes are found in loops around the active site which open the narrow active site channel of AT to form an open and solvent-exposed cavity. In particular, module II of StrB1 is AT-like but lacks a 10-residue α-helix in the 170-loop....
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Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
European journal of biochemistry, 1997Co-Authors: Erich Fritsche, Andreas Humm, Robert HuberAbstract:L-Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active-site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recom-binant human L-arginine : glycine amidinotransferase were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28-nm, 0.29-nm and 0.236-nm resolution, respectively. The mutation of active-site residues which are involved in substrate-binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000-fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild-type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition-state stabilization, and not due to major conformational rearrangements of the protein. These results show that a His-Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain-like cysteine proteinases.
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CRYSTAL STRUCTURE AND MECHANISM OF HUMAN L-ARGININE: GLYCINE AMIDINOTRANSFERASE: A MITOCHONDRIAL ENZYME INVOLVED IN CREATINE BIOSYNTHESIS
The EMBO journal, 1997Co-Authors: Andreas Humm, Erich Fritsche, Stefan Steinbacher, Robert HuberAbstract:L-arginine:glycine amidinotransferase (AT) catalyses the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the immediate precursor of creatine. We have determined the crystal structure of the recombinant human enzyme by multiple isomorphous replacement at 1.9 A resolution. A telluromethionine derivative was used in sequence assignment. The structure of AT reveals a new fold with 5-fold pseudosymmetry of circularly arranged betabeta alphabeta-modules. These enclose the active site compartment, which is accessible only through a narrow channel. The overall structure resembles a basket with handles that are formed from insertions into the betabeta alphabeta-modules. Binding of L-ornithine, a product inhibitor, reveals a marked induced-fit mechanism, with a loop at the active site entrance changing its conformation accompanied by a shift of an alpha-helix by -4 A. Binding of the arginine educt to the inactive mutant C407A shows a similar mode of binding. A reaction mechanism with a catalytic triad Cys-His-Asp is proposed on the basis of substrate and product bound states.
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recombinant expression and isolation of human l arginine glycine amidinotransferase and identification of its active site cysteine residue
Biochemical Journal, 1997Co-Authors: Andreas Humm, Erich Fritsche, Karlheinz Mann, Martin Gohl, Robert HuberAbstract:Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine:glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coli with two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.
Aaron Kaplan - One of the best experts on this subject based on the ideXlab platform.
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an abrb like protein might be involved in the regulation of cylindrospermopsin production by aphanizomenon ovalisporum
Environmental Microbiology, 2008Co-Authors: Gali Shalevmalul, Judy Liemanhurwitz, Yehudith Vinermozzini, Ariel Gaathon, Mario Lebendiker, Assaf Sukenik, Aaron KaplanAbstract:Certain filamentous cyanobacteria, including Aphanizomenon ovalisporum, are potentially toxic owing to the formation of the hepatotoxin cylindrospermopsin. We previously identified a gene cluster in A. ovalisporum likely to be involved in cylindrospermopsin biosynthesis, including amidinotransferase (aoaA) and polyketide-synthase (aoaC), transcribed on the reverse strands. Analysis of the genomic region between aoaA and aoaC identified two transcription start points for each of these genes, differentially expressed under nitrogen and light stress conditions. The transcript abundances of these genes and the cylindrospermopsin level were both affected by nitrogen availability and light intensity. Gel shift assays and DNA affinity columns isolated a protein that specifically binds to a 150 bp DNA fragment from the region between aoaA and aoaC, and MS/MS analyses identified similarity to AbrB in other cyanobacteria and in Bacillus sp. Comparison of the native AbrB isolated from A. ovalisporum with that obtained after cloning and overexpression of abrB in Escherichia coli identified specific post-translational modifications in the native cyanobacterial protein. These modifications, which are missing in the protein expressed in E. coli, include N-acetylation and methylation of specific residues. We discuss the possible role of these modifications in the regulation of cylindrospermopsin production in Aphanizomenon.
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A novel gene encoding amidinotransferase in the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum
Fems Microbiology Letters, 2002Co-Authors: Gali Shalev-alon, Oded Livnah, Rakefet Schwarz, Assaf Sukenik, Aaron KaplanAbstract:The hepatotoxin cylindrospermopsin is produced by several cyanobacteria species, which may flourish in tropical and sub-tropical lakes. Biosynthesis of cylindrospermopsin is poorly understood but its chemical nature, and feeding experiments with stable isotopes, suggested that guanidinoacetic acid is the starter unit and indicated involvement of a polyketide synthase. We have identified a gene encoding an amidinotransferase from the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum. This is the first report on an amidinotransferase gene in cyanobacteria. It is likely to be involved in the formation of guanidinoacetic acid. The aoaA is located in a genomic region bearing genes encoding a polyketide synthase and a peptide synthetase, further supporting its putative role in cylindrospermopsin biosynthesis.
Ariel Alvarez-morales - One of the best experts on this subject based on the ideXlab platform.
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Isolation and Characterization of the Gene Coding for the Amidinotransferase Involved in the Biosynthesis of Phaseolotoxin in Pseudomonas syringae pv. phaseolicola
Molecular plant-microbe interactions : MPMI, 2001Co-Authors: Gustavo Hernández-guzmán, Ariel Alvarez-moralesAbstract:Pseudomonas syringae pv. phaseolicola is the causal agent of the “halo blight” disease of beans. A key component in the development of the disease is a nonhost-specific toxin, Nδ-(N'-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine, known as phaseolotoxin. The homoarginine residue in this molecule has been suggested to be the product of Larginine:lysine amidinotransferase activity, previously detected in extracts of P. syringae pv. phaseolicola grown under conditions of phaseolotoxin production. We report the isolation and characterization of an amidinotransferase gene (amtA) from P. syringae pv. phaseolicola coding for a polypeptide of 362 residues (41.36 kDa) and showing approximately 40% sequence similarity to Larginine:inosamine-phosphate amidinotransferase from three species of Streptomyces spp. and 50.4% with an Larginine:glycine amidinotransferase from human mitochondria. The cysteine, histidine, and aspartic acid residues involved in substrate binding are conserved. Furthermore, expression of...