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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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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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substrate binding and catalysis by l arginine Glycine Amidinotransferase a mutagenesis and crystallographic study
FEBS Journal, 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.
Chi-un Choe - One of the best experts on this subject based on the ideXlab platform.
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analysis of l arginine Glycine Amidinotransferase creatine and homoarginine dependent gene regulation in the murine heart
Scientific Reports, 2020Co-Authors: Marit Jensen, Chi-un Choe, Edzard Schwedhelm, Christian P Muller, Tanja ZellerAbstract:L-arginine:Glycine Amidinotransferase (AGAT) and its metabolites creatine and homoarginine (HA) have been linked to cardiovascular pathologies in both human and murine studies, but the underlying molecular mechanisms are poorly understood. Here, we report the first analysis of heart transcriptome variation using microarrays in an AGAT-deficient (AGAT-/-) mouse model to evaluate AGAT-, creatine- and HA-dependent gene regulation. Our data revealed significant differences of gene expression between AGAT-/- and wild-type (WT) mice, affecting cardiac energy metabolism (Fbp2, Ucp2), cardiac hypertrophy and fibrosis (Nppa, Ctgf), immune response (Fgl2), and the conduction system of the heart (Dsc2, Ehd4, Hcn2, Hcn4, Scn4a, Scn4b). All of these genes being expressed on WT level in creatine-supplemented mice. Using in silico analysis based on the GEO database we found that most of these candidate genes (Ctgf, Dsc2, Fbp2, Fgl2, Hcn2, Nppa) revealed significant alterations in a WT mouse model of myocardial infarction underlining a pathophysiological relationship between AGAT metabolism and cardiovascular disease.
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homoarginine and creatine dependent gene regulation in murine brains with l arginine Glycine Amidinotransferase deficiency
International Journal of Molecular Sciences, 2020Co-Authors: Marit Jensen, Edzard Schwedhelm, Christian P Muller, Priyadharshini Arunachalam, Mathias Gelderblom, Tim Magnus, Christian Gerloff, Tanja Zeller, Chi-un ChoeAbstract:l-arginine:Glycine Amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to stroke pathology in both human and mouse studies. However, a comprehensive understanding of the underlying molecular mechanism is lacking. To investigate transcriptional changes in cerebral AGAT metabolism, we applied a transcriptome analysis in brains of wild-type (WT) mice compared to untreated AGAT-deficient (AGAT-/-) mice and AGAT-/- mice with creatine or hArg supplementation. We identified significantly regulated genes between AGAT-/- and WT mice in two independent cohorts of mice which can be linked to amino acid metabolism (Ivd, Lcmt2), creatine metabolism (Slc6a8), cerebral myelination (Bcas1) and neuronal excitability (Kcnip3). While Ivd and Kcnip3 showed regulation by hArg supplementation, Bcas1 and Slc6a8 were creatine dependent. Additional regulated genes such as Pla2g4e and Exd1 need further evaluation of their influence on cerebral function. Experimental stroke models showed a significant regulation of Bcas1 and Slc6a8. Together, these results reveal that AGAT deficiency, hArg and creatine regulate gene expression in the brain, which may be critical in stroke pathology.
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impaired cardiac contractile function in arginine Glycine Amidinotransferase knockout mice devoid of creatine is rescued by homoarginine but not creatine
Cardiovascular Research, 2018Co-Authors: Kiterie M E Faller, Dorothee Atzler, Hannah J Whittington, Sevasti Zervou, Debra J Mcandrew, Jillian N Simon, Dunja Aksentijevic, Michiel Ten Hove, Chi-un ChoeAbstract:Aims: Creatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:Glycine Amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality. Methods and results: Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naive to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naive AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function. Conclusions: Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases.
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homoarginine and l arginine Glycine Amidinotransferase in stroke
2017Co-Authors: Chi-un Choe, Edzard Schwedhelm, Dorothee AtzlerAbstract:Stroke is the second most frequent cause of death and leading cause of disability worldwide [Donnan et al. (Lancet 371:1612–1623, 2008)]. A third of stroke patients die within 1 year and more than half of the stroke patients are dead after 5 years [Hankey et al. (Stroke 29:2491–2500, 1998; Stroke 31:2080–2086, 2000)]. The most common causes of death are recurrent stroke and cardiovascular disease. In general, large and small vessel arteriosclerosis underlies both vascular entities. Consequently, cardiovascular disease and stroke pathology involve the same vascular risk factors. Therapeutic strategies and research have focused especially on modifiable risk factors, i.e., hypertension, diabetes, hyperlipidemia, and obesity. The combination of these four risk factors is known as metabolic syndrome, which is the consequence of supernutrition and excess body fat. Interestingly, reduced body weight is also associated with vascular mortality eliciting a bell-shaped association of body mass index (BMI) with vascular death [Chen et al. (BMJ 347:f5446, 2013); Hankey (Lancet Neurol 11:66–81, 2012)]. Therefore, balanced caloric intake and expenditure are necessary to optimize nutrition and health. In addition to established and conventional metabolic risk factors, much effort has been spent to identify novel metabolic pathways, which could influence vascular pathology and outcome. The long known, but until recently neglected, endogenous amino acid homoarginine has emerged as a significant marker and even more importantly as potential mediator of vascular disease [Choe et al. (Circulation 128:1451–1461, 2013); Marz et al. (Circulation 122:967–975, 2010)]. The enzyme, which catalyzes the synthesis of homoarginine, is l-arginine/Glycine Amidinotransferase (AGAT, EC 2.1.4.1). So far, AGAT was only known as the first and rate-limiting enzyme of creatine synthesis. To understand the role of AGAT in stroke pathology, it is mandatory to differentiate effects of AGAT metabolites creatine and homoarginine.
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Homoarginine and l-Arginine/Glycine Amidinotransferase in Stroke
L-Arginine in Clinical Nutrition, 2016Co-Authors: Chi-un Choe, Edzard Schwedhelm, Dorothee AtzlerAbstract:Stroke is the second most frequent cause of death and leading cause of disability worldwide [Donnan et al. (Lancet 371:1612–1623, 2008)]. A third of stroke patients die within 1 year and more than half of the stroke patients are dead after 5 years [Hankey et al. (Stroke 29:2491–2500, 1998; Stroke 31:2080–2086, 2000)]. The most common causes of death are recurrent stroke and cardiovascular disease. In general, large and small vessel arteriosclerosis underlies both vascular entities. Consequently, cardiovascular disease and stroke pathology involve the same vascular risk factors. Therapeutic strategies and research have focused especially on modifiable risk factors, i.e., hypertension, diabetes, hyperlipidemia, and obesity. The combination of these four risk factors is known as metabolic syndrome, which is the consequence of supernutrition and excess body fat. Interestingly, reduced body weight is also associated with vascular mortality eliciting a bell-shaped association of body mass index (BMI) with vascular death [Chen et al. (BMJ 347:f5446, 2013); Hankey (Lancet Neurol 11:66–81, 2012)]. Therefore, balanced caloric intake and expenditure are necessary to optimize nutrition and health. In addition to established and conventional metabolic risk factors, much effort has been spent to identify novel metabolic pathways, which could influence vascular pathology and outcome. The long known, but until recently neglected, endogenous amino acid homoarginine has emerged as a significant marker and even more importantly as potential mediator of vascular disease [Choe et al. (Circulation 128:1451–1461, 2013); Marz et al. (Circulation 122:967–975, 2010)]. The enzyme, which catalyzes the synthesis of homoarginine, is l-arginine/Glycine Amidinotransferase (AGAT, EC 2.1.4.1). So far, AGAT was only known as the first and rate-limiting enzyme of creatine synthesis. To understand the role of AGAT in stroke pathology, it is mandatory to differentiate effects of AGAT metabolites creatine and homoarginine.
Munir Pirmohamed - One of the best experts on this subject based on the ideXlab platform.
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GATM gene variants and statin myopathy risk
Nature, 2014Co-Authors: Daniel F. Carr, Ana Alfirevic, R Johnson, Hector Chinoy, T. Van Staa, Munir PirmohamedAbstract:Arising from L. M. Mangravite et al . Nature 502, 377–380 (2013); doi:10.1038/nature12508 Mangravite et al. ^ 1 identified six expression quantitative loci (eQTLs) that interacted with simvastatin exposure by using 480 lymphoblastoid cell lines exposed to β-hydroxy simvastatin acid in vitro . One of these SNPs (rs9806699) within the Glycine Amidinotransferase ( GATM ) gene was shown to have an association with statin-induced myopathy in two independent cohorts ( n = 172 myopathy cases), conferring a protective effect (odds ratio = 0.61, 95% confidence interval = 0.39–0.95, P = 0.03). Our genotyping results from statin myopathy patients do not appear to replicate this finding. There is a Reply to this Brief Communication Arising by Mangravite, L. M. et al. Nature 513, http://dx.doi.org/10.1038/nature13630 (2014).
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GATM gene variants and statin myopathy risk
Nature, 2014Co-Authors: Daniel F. Carr, Ana Alfirevic, R Johnson, Hector Chinoy, T. P. Van Staa, Munir PirmohamedAbstract:Arising from L. M. Mangravite . 377–380 (2013); doi:10.1038/nature12508 Mangravite et al.1 identified six expression quantitative loci (eQTLs) that interacted with simvastatin exposure by using 480 lymphoblastoid cell lines exposed to β-hydroxy simvastatin acid in vitro. One of these SNPs (rs9806699) within the Glycine Amidinotransferase (GATM) gene was shown to have an association with statin-induced myopathy in two independent cohorts (n = 172 myopathy cases), conferring a protective effect (odds ratio = 0.61, 95% confidence interval = 0.39–0.95, P = 0.03). Our genotyping results from statin myopathy patients do not appear to replicate this finding. There is a Reply to this Brief Communication Arising by Mangravite, L. M. et al. Nature 513, http://dx.doi.org/10.1038/nature13630 (2014).
Sonia Nouioua - One of the best experts on this subject based on the ideXlab platform.
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arginine Glycine Amidinotransferase agat deficiency clinical features and long term outcomes in 16 patients diagnosed worldwide
Molecular Genetics and Metabolism, 2015Co-Authors: Sylvia Stockleripsiroglu, Roberta Battini, Delia Apatean, Suzanne D Debrosse, Kimberley Dessoffy, Simon Edvardson, Florian Eichler, Katherine Johnston, David M Koeller, Sonia NouiouaAbstract:Abstract Background Arginine:Glycine aminotransferase (AGAT) (GATM) deficiency is an autosomal recessive inborn error of creative synthesis. Objective We performed an international survey among physicians known to treat patients with AGAT deficiency, to assess clinical characteristics and long-term outcomes of this ultra-rare condition. Results 16 patients from 8 families of 8 different ethnic backgrounds were included. 1 patient was asymptomatic when diagnosed at age 3 weeks. 15 patients diagnosed between 16 months and 25 years of life had intellectual disability/developmental delay (IDD). 8 patients also had myopathy/proximal muscle weakness. Common biochemical denominators were low/undetectable guanidinoacetate (GAA) concentrations in urine and plasma, and low/undetectable cerebral creatine levels. 3 families had protein truncation/null mutations. The rest had missense and splice mutations. Treatment with creatine monohydrate (100–800 mg/kg/day) resulted in almost complete restoration of brain creatine levels and significant improvement of myopathy. The 2 patients treated since age 4 and 16 months had normal cognitive and behavioral development at age 10 and 11 years. Late treated patients had limited improvement of cognitive functions. Conclusion AGAT deficiency is a treatable intellectual disability. Early diagnosis may prevent IDD and myopathy. Patients with unexplained IDD with and without myopathy should be assessed for AGAT deficiency by determination of urine/plasma GAA and cerebral creatine levels (via brain MRS), and by GATM gene sequencing.
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creatine deficiency syndrome a treatable myopathy due to arginine Glycine Amidinotransferase agat deficiency
Neuromuscular Disorders, 2013Co-Authors: Sonia Nouioua, D. Cheillan, S. Zaouidi, G.s. Salomons, N. Amedjout, F. Kessaci, N. Boulahdour, Tarik Hamadouche, Meriem TazirAbstract:Abstract We report two sisters, aged 11 and 6years, with AGAT deficiency syndrome (OMIM 612718) which is the least common creatine deficiency syndrome. They were born full-term to consanguineous parents and had moderate developmental delay. Examination showed an important language delay, a progressive proximal muscular weakness in the lower limbs with Gowers sign and myopathic electromyography. Investigations revealed undetectable guanidinoacetate and low level of creatine in plasma and urine, characteristic findings of AGAT deficiency syndrome. Brain magnetic resonance spectroscopy showed a markedly reduced level of creatine. Guanidinoacetate methyltransferase ( GATM ) gene sequencing revealed a homozygous missense mutation in exon 4:c.608A>C, (p.Tyr203Ser). Thirteen months after beginning the treatment with oral creatine monohydrate 200mg/kg/day, then 400mg/kg/day, there was a dramatic improvement in muscle strength with Gowers sign disappearance in both patients, and a mild improvement in language and cognitive functions. AGAT deficiency syndrome should be considered in all patients with language retardation and cognitive impairment associated to a myopathy of unknown etiology such that early diagnosis must lead to creatine supplementation to cure the myopathy and improve language and cognitive function.
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Creatine deficiency syndrome. A treatable myopathy due to arginine–Glycine Amidinotransferase (AGAT) deficiency
Neuromuscular disorders : NMD, 2013Co-Authors: Sonia Nouioua, D. Cheillan, S. Zaouidi, G.s. Salomons, N. Amedjout, F. Kessaci, N. Boulahdour, Tarik Hamadouche, Meriem TazirAbstract:Abstract We report two sisters, aged 11 and 6years, with AGAT deficiency syndrome (OMIM 612718) which is the least common creatine deficiency syndrome. They were born full-term to consanguineous parents and had moderate developmental delay. Examination showed an important language delay, a progressive proximal muscular weakness in the lower limbs with Gowers sign and myopathic electromyography. Investigations revealed undetectable guanidinoacetate and low level of creatine in plasma and urine, characteristic findings of AGAT deficiency syndrome. Brain magnetic resonance spectroscopy showed a markedly reduced level of creatine. Guanidinoacetate methyltransferase ( GATM ) gene sequencing revealed a homozygous missense mutation in exon 4:c.608A>C, (p.Tyr203Ser). Thirteen months after beginning the treatment with oral creatine monohydrate 200mg/kg/day, then 400mg/kg/day, there was a dramatic improvement in muscle strength with Gowers sign disappearance in both patients, and a mild improvement in language and cognitive functions. AGAT deficiency syndrome should be considered in all patients with language retardation and cognitive impairment associated to a myopathy of unknown etiology such that early diagnosis must lead to creatine supplementation to cure the myopathy and improve language and cognitive function.
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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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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substrate binding and catalysis by l arginine Glycine Amidinotransferase a mutagenesis and crystallographic study
FEBS Journal, 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.