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Amidinotransferases

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Robert Huber – One of the best experts on this subject based on the ideXlab platform.

  • the ligand induced structural changes of human l arginine glycine amidinotransferase a mutational and crystallographic study
    Journal of Biological Chemistry, 1999
    Co-Authors: Erich Fritsche, Andreas Humm, Robert Huber
    Abstract:

    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.

  • Crystal Structure of l-Arginine:Inosamine-Phosphate Amidinotransferase StrB1 from Streptomyces griseus: An Enzyme Involved in Streptomycin Biosynthesis‡
    Biochemistry, 1998
    Co-Authors: Erich Fritsche, Andreas Humm, Andreas Bergner, Wolfgang Piepersberg, Robert Huber
    Abstract:

    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….

  • Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
    European journal of biochemistry, 1997
    Co-Authors: Erich Fritsche, Andreas Humm, Robert Huber
    Abstract:

    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 mutamutagenesis 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.

Andreas Humm – One of the best experts on this subject based on the ideXlab platform.

  • the ligand induced structural changes of human l arginine glycine amidinotransferase a mutational and crystallographic study
    Journal of Biological Chemistry, 1999
    Co-Authors: Erich Fritsche, Andreas Humm, Robert Huber
    Abstract:

    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.

  • Crystal Structure of l-Arginine:Inosamine-Phosphate Amidinotransferase StrB1 from Streptomyces griseus: An Enzyme Involved in Streptomycin Biosynthesis‡
    Biochemistry, 1998
    Co-Authors: Erich Fritsche, Andreas Humm, Andreas Bergner, Wolfgang Piepersberg, Robert Huber
    Abstract:

    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….

  • Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
    European journal of biochemistry, 1997
    Co-Authors: Erich Fritsche, Andreas Humm, Robert Huber
    Abstract:

    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.

Erich Fritsche – One of the best experts on this subject based on the ideXlab platform.

  • the ligand induced structural changes of human l arginine glycine amidinotransferase a mutational and crystallographic study
    Journal of Biological Chemistry, 1999
    Co-Authors: Erich Fritsche, Andreas Humm, Robert Huber
    Abstract:

    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.

  • Crystal Structure of l-Arginine:Inosamine-Phosphate Amidinotransferase StrB1 from Streptomyces griseus: An Enzyme Involved in Streptomycin Biosynthesis‡
    Biochemistry, 1998
    Co-Authors: Erich Fritsche, Andreas Humm, Andreas Bergner, Wolfgang Piepersberg, Robert Huber
    Abstract:

    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….

  • Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
    European journal of biochemistry, 1997
    Co-Authors: Erich Fritsche, Andreas Humm, Robert Huber
    Abstract:

    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.

Aaron Kaplan – One of the best experts on this subject based on the ideXlab platform.

  • an abrb like protein might be involved in the regulation of cylindrospermopsin production by aphanizomenon ovalisporum
    Environmental Microbiology, 2008
    Co-Authors: Gali Shalevmalul, Assaf Sukenik, Judy Liemanhurwitz, Yehudith Vinermozzini, Ariel Gaathon, Mario Lebendiker, Aaron Kaplan
    Abstract:

    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.

  • A novel gene encoding amidinotransferase in the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum
    Fems Microbiology Letters, 2002
    Co-Authors: Gali Shalev-alon, Assaf Sukenik, Oded Livnah, Rakefet Schwarz, Aaron Kaplan
    Abstract:

    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.

  • 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, 2001
    Co-Authors: Gustavo Hernández-guzmán, Ariel Alvarez-morales
    Abstract:

    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…