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Motoji Fujioka - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism of Guanidinoacetate Methyltransferase crystal structures of Guanidinoacetate Methyltransferase ternary complexes
Biochemistry, 2004Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. The intact GAMT from recombinant rat liver has been crystallized with an inhibitor S-adenosylhomocysteine (SAH) and a substrate Guanidinoacetate (GAA), and with SAH and an inhibitor guanidine (GUN). These ternary complex structures have been determined at 2.0 A resolution. GAMT has an α/β open-sandwich structure, and the N-terminal section (residues 1−42) covers the active site entrance so that the active site is not visible. SAH has extensive interactions with GAMT through H-bonds and hydrophobic interactions. The guanidino groups of GAA and GUN form two pairs of H-bonds with E45 and D134, respectively. The carboxylate group of GAA interacts with the backbone amide groups of L170 and T171. A model structure of GAMT containing the two substrates (SAM and GAA) was built by attaching a methyl group (CE) on SD of the bound SAH. On th...
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monoclinic Guanidinoacetate Methyltransferase and gadolinium ion binding characteristics
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Fusao TakusagawaAbstract:: Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT truncated at amino acid 37 from the N-terminus has been crystallized with S-adenosylhomocysteine (SAH) in a monoclinic modification and the crystal structure has been determined at 2.8 A resolution. There are two dimers in the crystallographic asymmetric unit. Each dimer has non-crystallographic twofold symmetry and is related to the other dimer by pseudo-4(3) symmetry along the crystallographic b axis. The overall structure of GAMT crystallized in the monoclinic modification is quite similar to the structure observed in the tetragonal modification [Komoto et al. (2002), J. Mol. Biol. 320, 223-235], with the exception of the loop containing Tyr136. In the monoclinic modification, the loops in three of the four subunits have a catalytically unfavorable conformation and the loop of the fourth subunit has a catalytically favorable conformation as observed in the crystals of the tetragonal modification. From the structures in the monoclinic and tetragonal modifications, we can explain why the Y136F mutant enzyme retains considerable catalytic activity while the Y136V mutant enzyme loses the catalytic activity. The crystal structure of a Gd derivative of the tetragonal modification has also been determined. By comparing the Gd-derivative structure with the native structures in the tetragonal and the monoclinic modifications, useful characteristic features of Gd-ion binding for application in protein crystallography have been observed. Gd ions can bind to proteins without changing the native protein structures and Gd atoms produce strong anomalous dispersion signals from Cu Kalpha radiation; however, Gd-ion binding to protein requires a relatively specific geometry.
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crystal structure of Guanidinoacetate Methyltransferase from rat liver a model structure of protein arginine Methyltransferase
Journal of Molecular Biology, 2002Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Yafei Huang, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the Guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine Methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine Methyltransferases.
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crystallization and preliminary x ray diffraction studies of Guanidinoacetate Methyltransferase from rat liver
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Yafei Huang, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase is the enzyme which catalyzes the last step of creatine biosynthesis. The enzyme is found ubiquitously and in abundance in the livers of all vertebrates. Recombinant rat-liver Guanidinoacetate Methyltransferase has been crystallized with Guanidinoacetate and S-adenosylhomocysteine. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.8, b = 162.5, c = 56.1 A, β = 96.8 (1)° at 93 K, and typically diffract beyond 2.8 A.
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probing the s adenosylmethionine binding site of rat Guanidinoacetate Methyltransferase effect of site directed mutagenesis of residues that are conserved across mammalian non nucleic acid Methyltransferases
Biochemical Journal, 1996Co-Authors: Akiko Hamahata, Yoshimi Takata, Tomoharu Gomi, Motoji FujiokaAbstract:Most mammalian non-nucleic acid Methyltransferases share three sequence motifs. To gain insight into the S -adenosylmethionine (AdoMet)-binding site of Guanidinoacetate Methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly 67 and Gly 69 ) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr 136 of motif II to a valine resulted in a decrease in k cat / K m values for substrates. Changing this residue to a phenylalanine caused only a minor change in k cat / K m for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu 89 , which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of Guanidinoacetate Methyltransferase.
I Antonozzi - One of the best experts on this subject based on the ideXlab platform.
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a mutation on exon 6 of Guanidinoacetate Methyltransferase gamt gene supports a different function for isoform a and b of gamt enzyme
Molecular Genetics and Metabolism, 2006Co-Authors: Vincenzo Leuzzi, Claudia Carducci, Maria Lucia Di Sabato, Carla Carducci, C Artiola, Marco Matricardi, M Bianchi, I AntonozziAbstract:A new patient affected by Guanidinoacetate Methyltransferase (GAMT) deficiency was reported. This 13-year-old girl presented with mental retardation, as main symptom, associated with a typical pattern of biochemical and neurochemical (brain magnetic resonance spectroscopy) alterations. Molecular study detected a L197P transition on exon 6 of the GAMT gene. Since this mutation leaves the isoform B of the GAMT enzyme unaffected, the occurrence of biochemical alterations and disease in this subject testifies against the possibility that isoform b had GAMT activity.
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Guanidinoacetate and creatine plus creatinine assessment in physiologic fluids an effective diagnostic tool for the biochemical diagnosis of arginine glycine amidinotransferase and Guanidinoacetate Methyltransferase deficiencies
Clinical Chemistry, 2002Co-Authors: Claudia Carducci, M Birarelli, Roberta Battini, Giovanni Cioni, Carla Carducci, Vincenzo Leuzzi, I AntonozziAbstract:Background: Disorders of creatine metabolism arise from genetic alterations of arginine:glycine amidinotransferase (AGAT), Guanidinoacetate Methyltransferase (GAMT), and the creatine transporter. We developed a strategy for the detection of AGAT and GAMT defects by measurement of Guanidinoacetate (GAA) and creatine plus creatinine (Cr+Crn) in biological fluids. Methods: Three patients with AGAT deficiency from the same pedigree and their eight relatives, as well as a patient affected by a GAMT defect and his parents were analyzed by a new HPLC procedure in comparison with 90 controls. The method, which uses precolumn derivatization with benzoin, separation with a reversed-phase column, and fluorescence detection, has shown good precision and sensitivity and requires minimal sample handling. Results: In the three AGAT patients, plasma GAA was 0.01–0.04 μmol/L [mean (SD) for neurologically normal controls was 1.16 (0.59) μmol/L], Cr+Crn was 15–29 μmol/L [reference limit in our laboratory, 79 (38) μmol/L]. Urinary GAA was 2.4–5.8 μmol/L [reference, 311 (191) μmol/L], and Cr+Crn was 2.1–3.3 mmol/L [reference, 9.9 (4.1) mmol/L]. We found a smaller decrease in GAA and Cr+Crn in some carriers of an AGAT defect. In the patient with GAMT deficiency, plasma and urine GAA was increased (18.6 and 1783 μmol/L, respectively), and Cr+Crn was decreased in plasma (10.7 μmol/L) and urine (2.1 mmol/L). GAA was increased in the parents’ plasmas and in the mother’s urine. Conclusion: The assessment of GAA is a new tool for the detection of both GAMT and AGAT deficiencies.
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automated high performance liquid chromatographic method for the determination of guanidinoacetic acid in dried blood spots a tool for early diagnosis of Guanidinoacetate Methyltransferase deficiency
Journal of Chromatography B: Biomedical Sciences and Applications, 2001Co-Authors: Claudia Carducci, M Birarelli, Pino Santagata, Carla Carducci, Vincenzo Leuzzi, I AntonozziAbstract:A new automated method for the assay of guanidinoacetic acid (GAA) in dried blood spot (DBS) on filter paper is reported. The method, based on reversed-phase (RP)-HPLC, precolumn derivatisation with benzoin and fluorescence detection, has shown good precision and sensitivity and requires only minimal sample handling. The validity of the method was demonstrated by analysing the neonatal blood spot of a patient affected by Guanidinoacetate Methyltransferase (GAMT) deficiency. GAA concentration was found to be nearly 12-fold higher than the mean control value. We propose this method as an inexpensive and widely applicable tool for the diagnosis of GAMT deficiency.
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two new severe mutations causing Guanidinoacetate Methyltransferase deficiency
Molecular Genetics and Metabolism, 2000Co-Authors: Carla Carducci, Claudia Carducci, Vincenzo Leuzzi, Sabrina Prudente, Luana Mercuri, I AntonozziAbstract:Abstract Primary disorders of creatine metabolism have been only recently described. We report new molecular and biochemical findings obtained from a child affected by Guanidinoacetate Methyltransferase deficiency. This patient presented with neurological regression, epilepsy, and a movement disorder during the first year of life. HPLC analysis showed high concentrations of guanidinoacetic acid in urine, plasma, and CSF. Molecular analyses of cDNA and genomic DNA revealed two novel mutations, a G insertion following nucleotide 491 of the cDNA (c.491insG) in exon 5 and a transversion at nt −3 in intron 5 (IVS5-3C>G). The c.491insG mutation causes a frameshift and a premature stop codon at the end of the exon. The IVS5-3C>G mutation prevents the splicing of the last exon of the gene precluding the complete maturation of the transcript and, most likely, causes rapid degradation of the mRNA.
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brain creatine depletion Guanidinoacetate Methyltransferase deficiency improving with creatine supplementation
Neurology, 2000Co-Authors: Vincenzo Leuzzi, M C Bianchi, Claudia Carducci, Antonella Cerquiglini, Giovanni Cioni, Michela Tosetti, I AntonozziAbstract:Article abstract The authors describe an Italian child with Guanidinoacetate Methyltransferase deficiency, neurologic regression, movement disorders, and epilepsy during the first year of life. Brain MRI showed pallidal and periaqueductal alterations. In vivo 1 H-MRS showed brain creatine depletion. The assessment of guanidinoacetic acid concentration in biologic fluids confirmed the diagnosis. Clinical, biochemical, and neuroradiologic improvement followed creatine supplementation.
Andreas Schulze - One of the best experts on this subject based on the ideXlab platform.
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elevation of Guanidinoacetate in newborn dried blood spots and impact of early treatment in gamt deficiency
Molecular Genetics and Metabolism, 2013Co-Authors: Areeg Elgharbawy, Amie E Vaisnins, Bruce Barshop, Dwight D. Koeberl, Jennifer L. Goldstein, Andreas Schulze, Andrea Schlune, Sarah P YoungAbstract:Abstract Guanidinoacetate Methyltransferase (GAMT) deficiency is a good candidate disorder for newborn screening because early treatment appears to improve outcomes. We report elevation of Guanidinoacetate in archived newborn dried blood spots for 3 cases (2 families) of GAMT deficiency compared with an unaffected carrier and controls. We also report a new case of a patient treated from birth with normal developmental outcome at the age of 42 months.
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successful treatment of a Guanidinoacetate Methyltransferase deficient patient findings with relevance to treatment strategy and pathophysiology
Molecular Genetics and Metabolism, 2007Co-Authors: K T Verbruggen, Roelinka J Lunsing, Andreas Schulze, Paul E. Sijens, Francjan J. SpronsenAbstract:Biochemical and developmental results of treatment of a Guanidinoacetate Methyltransferase (GAMT) deficient patient with a mild clinical presentation and remarkable developmental improvement after treatment are presented. Treatment with creatine (Cr) supplementation resulted in partial normalization of cerebral (measured with magnetic resonance proton spectroscopy) and plasma levels of Cr and Guanidinoacetate (GAA). Addition of high dose ornithine to the treatment led to further normalization of plasma GAA, while cerebral Cr and GAA did not improve further.
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presymptomatic treatment of neonatal Guanidinoacetate Methyltransferase deficiency
Neurology, 2006Co-Authors: Andreas Schulze, Peter Bachert, Stefan Kirsch, N M Verhoeven, Georg F Hoffmann, Ertan MayatepekAbstract:Prospective observation in a neonate with Guanidinoacetate Methyltransferase deficiency (GAMT-D), a severe neurometabolic disorder, revealed increased Guanidinoacetate levels at birth. After 14-month treatment with creatine, high-dose ornithine, benzoate, and an arginine-restricted diet, the patient9s development is normal and she does not present any symptoms of GAMT-D. The authors9 observation indicates that early detection of GAMT-D is possible in the neonatal period, and presymptomatic treatment may prevent its manifestation.
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Guanidinoacetate Methyltransferase deficiency differences of creatine uptake in human brain and muscle
Molecular Genetics and Metabolism, 2004Co-Authors: Karl Otfried Schwab, Ulrich Tacke, Silvia Stockleripsiroglu, Thorsten Thiel, Regina Ensenauer, Andreas Schulze, Jurgen Hennig, Wendy LehnertAbstract:Abstract Deficiency of Guanidinoacetate Methyltransferase (GAMT), the first described creatine biosynthesis defect, leads to depletion of creatine and phosphocreatine, and accumulation of Guanidinoacetate in brain. This results in epilepsy, mental retardation, and extrapyramidal movement disorders. Investigation of skeletal muscle by proton and phosphorus magnetic resonance spectroscopy before therapy demonstrated the presence of considerable amounts of creatine and phosphocreatine, and accumulation of phosphorylated Guanidinoacetate in a 7-year-old boy diagnosed with GAMT deficiency, suggesting separate mechanisms for creatine uptake and synthesis in brain and skeletal muscle. The combination of creatine supplementation and a Guanidinoacetate-lowering therapeutic approach resulted in improvement of clinical symptoms and metabolite concentrations in brain, muscle, and body fluids.
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creatine deficiency syndromes
Molecular and Cellular Biochemistry, 2003Co-Authors: Andreas SchulzeAbstract:Since the first description of a creatine deficiency syndrome, the Guanidinoacetate Methyltransferase (GAMT) deficiency, in 1994, the two further suspected creatine deficiency syndromes – the creatine transporter (CrT1) defect and the arginine:glycine amidinotransferase (AGAT) deficiency were disclosed.
Yoshimi Takata - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism of Guanidinoacetate Methyltransferase crystal structures of Guanidinoacetate Methyltransferase ternary complexes
Biochemistry, 2004Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. The intact GAMT from recombinant rat liver has been crystallized with an inhibitor S-adenosylhomocysteine (SAH) and a substrate Guanidinoacetate (GAA), and with SAH and an inhibitor guanidine (GUN). These ternary complex structures have been determined at 2.0 A resolution. GAMT has an α/β open-sandwich structure, and the N-terminal section (residues 1−42) covers the active site entrance so that the active site is not visible. SAH has extensive interactions with GAMT through H-bonds and hydrophobic interactions. The guanidino groups of GAA and GUN form two pairs of H-bonds with E45 and D134, respectively. The carboxylate group of GAA interacts with the backbone amide groups of L170 and T171. A model structure of GAMT containing the two substrates (SAM and GAA) was built by attaching a methyl group (CE) on SD of the bound SAH. On th...
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monoclinic Guanidinoacetate Methyltransferase and gadolinium ion binding characteristics
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Fusao TakusagawaAbstract:: Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT truncated at amino acid 37 from the N-terminus has been crystallized with S-adenosylhomocysteine (SAH) in a monoclinic modification and the crystal structure has been determined at 2.8 A resolution. There are two dimers in the crystallographic asymmetric unit. Each dimer has non-crystallographic twofold symmetry and is related to the other dimer by pseudo-4(3) symmetry along the crystallographic b axis. The overall structure of GAMT crystallized in the monoclinic modification is quite similar to the structure observed in the tetragonal modification [Komoto et al. (2002), J. Mol. Biol. 320, 223-235], with the exception of the loop containing Tyr136. In the monoclinic modification, the loops in three of the four subunits have a catalytically unfavorable conformation and the loop of the fourth subunit has a catalytically favorable conformation as observed in the crystals of the tetragonal modification. From the structures in the monoclinic and tetragonal modifications, we can explain why the Y136F mutant enzyme retains considerable catalytic activity while the Y136V mutant enzyme loses the catalytic activity. The crystal structure of a Gd derivative of the tetragonal modification has also been determined. By comparing the Gd-derivative structure with the native structures in the tetragonal and the monoclinic modifications, useful characteristic features of Gd-ion binding for application in protein crystallography have been observed. Gd ions can bind to proteins without changing the native protein structures and Gd atoms produce strong anomalous dispersion signals from Cu Kalpha radiation; however, Gd-ion binding to protein requires a relatively specific geometry.
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crystal structure of Guanidinoacetate Methyltransferase from rat liver a model structure of protein arginine Methyltransferase
Journal of Molecular Biology, 2002Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Yafei Huang, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the Guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine Methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine Methyltransferases.
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crystallization and preliminary x ray diffraction studies of Guanidinoacetate Methyltransferase from rat liver
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Yafei Huang, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase is the enzyme which catalyzes the last step of creatine biosynthesis. The enzyme is found ubiquitously and in abundance in the livers of all vertebrates. Recombinant rat-liver Guanidinoacetate Methyltransferase has been crystallized with Guanidinoacetate and S-adenosylhomocysteine. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.8, b = 162.5, c = 56.1 A, β = 96.8 (1)° at 93 K, and typically diffract beyond 2.8 A.
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probing the s adenosylmethionine binding site of rat Guanidinoacetate Methyltransferase effect of site directed mutagenesis of residues that are conserved across mammalian non nucleic acid Methyltransferases
Biochemical Journal, 1996Co-Authors: Akiko Hamahata, Yoshimi Takata, Tomoharu Gomi, Motoji FujiokaAbstract:Most mammalian non-nucleic acid Methyltransferases share three sequence motifs. To gain insight into the S -adenosylmethionine (AdoMet)-binding site of Guanidinoacetate Methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly 67 and Gly 69 ) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr 136 of motif II to a valine resulted in a decrease in k cat / K m values for substrates. Changing this residue to a phenylalanine caused only a minor change in k cat / K m for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu 89 , which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of Guanidinoacetate Methyltransferase.
Kiyoshi Konishi - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism of Guanidinoacetate Methyltransferase crystal structures of Guanidinoacetate Methyltransferase ternary complexes
Biochemistry, 2004Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. The intact GAMT from recombinant rat liver has been crystallized with an inhibitor S-adenosylhomocysteine (SAH) and a substrate Guanidinoacetate (GAA), and with SAH and an inhibitor guanidine (GUN). These ternary complex structures have been determined at 2.0 A resolution. GAMT has an α/β open-sandwich structure, and the N-terminal section (residues 1−42) covers the active site entrance so that the active site is not visible. SAH has extensive interactions with GAMT through H-bonds and hydrophobic interactions. The guanidino groups of GAA and GUN form two pairs of H-bonds with E45 and D134, respectively. The carboxylate group of GAA interacts with the backbone amide groups of L170 and T171. A model structure of GAMT containing the two substrates (SAM and GAA) was built by attaching a methyl group (CE) on SD of the bound SAH. On th...
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monoclinic Guanidinoacetate Methyltransferase and gadolinium ion binding characteristics
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Fusao TakusagawaAbstract:: Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT truncated at amino acid 37 from the N-terminus has been crystallized with S-adenosylhomocysteine (SAH) in a monoclinic modification and the crystal structure has been determined at 2.8 A resolution. There are two dimers in the crystallographic asymmetric unit. Each dimer has non-crystallographic twofold symmetry and is related to the other dimer by pseudo-4(3) symmetry along the crystallographic b axis. The overall structure of GAMT crystallized in the monoclinic modification is quite similar to the structure observed in the tetragonal modification [Komoto et al. (2002), J. Mol. Biol. 320, 223-235], with the exception of the loop containing Tyr136. In the monoclinic modification, the loops in three of the four subunits have a catalytically unfavorable conformation and the loop of the fourth subunit has a catalytically favorable conformation as observed in the crystals of the tetragonal modification. From the structures in the monoclinic and tetragonal modifications, we can explain why the Y136F mutant enzyme retains considerable catalytic activity while the Y136V mutant enzyme loses the catalytic activity. The crystal structure of a Gd derivative of the tetragonal modification has also been determined. By comparing the Gd-derivative structure with the native structures in the tetragonal and the monoclinic modifications, useful characteristic features of Gd-ion binding for application in protein crystallography have been observed. Gd ions can bind to proteins without changing the native protein structures and Gd atoms produce strong anomalous dispersion signals from Cu Kalpha radiation; however, Gd-ion binding to protein requires a relatively specific geometry.
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crystal structure of Guanidinoacetate Methyltransferase from rat liver a model structure of protein arginine Methyltransferase
Journal of Molecular Biology, 2002Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Taro Yamada, Yafei Huang, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the Guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine Methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine Methyltransferases.
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crystallization and preliminary x ray diffraction studies of Guanidinoacetate Methyltransferase from rat liver
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Junichi Komoto, Yoshimi Takata, Tomoharu Gomi, Motoji Fujioka, Kiyoshi Konishi, Hirofumi Ogawa, Yafei Huang, Fusao TakusagawaAbstract:Guanidinoacetate Methyltransferase is the enzyme which catalyzes the last step of creatine biosynthesis. The enzyme is found ubiquitously and in abundance in the livers of all vertebrates. Recombinant rat-liver Guanidinoacetate Methyltransferase has been crystallized with Guanidinoacetate and S-adenosylhomocysteine. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.8, b = 162.5, c = 56.1 A, β = 96.8 (1)° at 93 K, and typically diffract beyond 2.8 A.
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rat Guanidinoacetate Methyltransferase effect of site directed alteration of an aspartic acid residue that is conserved across most mammalian s adenosylmethionine dependent Methyltransferases
Journal of Biological Chemistry, 1994Co-Authors: Yoshimi Takata, Kiyoshi Konishi, Takamasa Gomi, M FujiokaAbstract:Abstract Most mammalian S-adenosylmethionine (AdoMet)-dependent Methyltransferases have a conserved aspartate residue in a sequence oDso (o denotes a hydrophobic amino acid and s denotes a small neutral amino acid). Rat Guanidinoacetate Methyltransferase has two aspartate residues (Asp-129 and Asp-134) conforming to the motif in close proximity to Tyr-136 that is photoaffinity-labeled by AdoMet (Takata, Y., and Fujioka, M. (1992) Biochemistry 31, 4369-4374). In order to investigate the role of these residues, we prepared variant forms of the enzyme by oligonucleotide-directed mutagenesis. Conversion of Asp-129 to asparagine or alanine resulted in a functional enzyme. Alteration of Asp-134 to glutamate (D134E) and asparagine (D134N) decreased activity, and replacement with alanine (D134A) led to inactivation. Decreases of 3- and 120-fold were found for kcat values of D134E and D134N, respectively. The Km values of D134E for AdoMet and those for Guanidinoacetate were increased about 160- and 80-fold over the respective values of wild type. The corresponding increases in D134N were 800- and 50-fold, respectively. Conservative changes of the residues flanking Asp-134 had little effect on activity. Guanidinoacetate Methyltransferase obeys an ordered Bi Bi mechanism in which AdoMet binds first. Thus, the large decreases in kcat/Km values for AdoMet indicate that Asp-134 is crucial for binding AdoMet. Spectroscopic studies indicated that the amino acid substitutions of Asp-134 resulted in no significant changes in the secondary and tertiary structures, and urea denaturation experiments showed that the altered enzymes were not destabilized.
