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Mendel Tuchman - One of the best experts on this subject based on the ideXlab platform.
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Gene delivery corrects N-Acetylglutamate Synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-Acetylglutamate Synthase.
Scientific reports, 2021Co-Authors: Parthasarathy Sonaimuthu, Hiroki Morizono, Mendel Tuchman, Nantaporn Haskins, Emilee Senkevitch, Prech Uapinyoying, Markey C. Mcnutt, Ljubica CaldovicAbstract:The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-Acetylglutamate Synthase (NAGS) catalyzes formation of N-Acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
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N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-Acetylglutamate Synthase Enhancer Region
Scientific reports, 2018Co-Authors: Monique Williams, Nantaporn Haskins, Alberto Burlina, Laura Rubert, Giulia Polo, George J. G. Ruijter, Myrthe Van Den Born, Véronique Rüfenacht, Laura J.c.m. Van Zutven, Mendel TuchmanAbstract:N-Acetylglutamate Synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-Acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD.
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Impaired ureagenesis due to arginine-insensitive N-Acetylglutamate Synthase
2018Co-Authors: Parthasarathy Sonaimuthu, Hiroki Morizono, Mendel Tuchman, Nantaporn Haskins, Emilee Senkevitch, Prech Uapinyoying, Markey C. Mcnutt, Ljubica CaldovicAbstract:The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to non-toxic urea. N-Acetylglutamate Synthase (NAGS) is an enzyme that catalyzes the formation of N-Acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1), the rate limiting enzyme of the urea cycle. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine but the physiological significance of NAGS activation by L-arginine is unknown. Previously, we have described the creation of a NAGS knockout (Nags-/-) mouse, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG+Cit). In order to investigate the effect of L-arginine on ureagenesis in vivo, we used adeno associated virus (AAV) mediated gene transfer to deliver either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine, to Nags-/- mice. The ability of the E354A mNAGS mutant protein to rescue Nags-/- mice was determined by measuring their activity on the voluntary wheel following NCG+Cit withdrawal. The Nags-/- mice that received E354A mNAGS remained apparently healthy and active but had elevated plasma ammonia concentration despite similar expression levels of the E354A mNAGS and control wild-type NAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was also identified in a patient with hyperammonemia due to NAGS deficiency. Taken together, our results suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
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Effect of arginine on oligomerization and stability of N-Acetylglutamate Synthase.
Scientific reports, 2016Co-Authors: Nantaporn Haskins, Hiroki Morizono, Mendel Tuchman, Amy Mumo, P H Brown, Ljubica CaldovicAbstract:N-Acetylglutamate Synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-Acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-Acetylglutamate Synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS.
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A novel UPLC-MS/MS based method to determine the activity of N-Acetylglutamate Synthase in liver tissue.
Molecular genetics and metabolism, 2016Co-Authors: M. Dercksen, A. Van Cruchten, Mendel Tuchman, Lodewijk Ijlst, Marinus Duran, Wim Kulik, Jos P.n. Ruiter, Ronald J A WandersAbstract:Abstract Background N -acetylglutamate Synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N -acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. Methods UPLC-MS/MS was used in conjunction with stable isotope ( N -acetylglutamic-2,3,3,4,4-d 5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. Results A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N -acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags −/− mice. Conclusion The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
Ljubica Caldovic - One of the best experts on this subject based on the ideXlab platform.
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Gene delivery corrects N-Acetylglutamate Synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-Acetylglutamate Synthase.
Scientific reports, 2021Co-Authors: Parthasarathy Sonaimuthu, Hiroki Morizono, Mendel Tuchman, Nantaporn Haskins, Emilee Senkevitch, Prech Uapinyoying, Markey C. Mcnutt, Ljubica CaldovicAbstract:The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-Acetylglutamate Synthase (NAGS) catalyzes formation of N-Acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
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Impaired ureagenesis due to arginine-insensitive N-Acetylglutamate Synthase
2018Co-Authors: Parthasarathy Sonaimuthu, Hiroki Morizono, Mendel Tuchman, Nantaporn Haskins, Emilee Senkevitch, Prech Uapinyoying, Markey C. Mcnutt, Ljubica CaldovicAbstract:The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to non-toxic urea. N-Acetylglutamate Synthase (NAGS) is an enzyme that catalyzes the formation of N-Acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1), the rate limiting enzyme of the urea cycle. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine but the physiological significance of NAGS activation by L-arginine is unknown. Previously, we have described the creation of a NAGS knockout (Nags-/-) mouse, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG+Cit). In order to investigate the effect of L-arginine on ureagenesis in vivo, we used adeno associated virus (AAV) mediated gene transfer to deliver either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine, to Nags-/- mice. The ability of the E354A mNAGS mutant protein to rescue Nags-/- mice was determined by measuring their activity on the voluntary wheel following NCG+Cit withdrawal. The Nags-/- mice that received E354A mNAGS remained apparently healthy and active but had elevated plasma ammonia concentration despite similar expression levels of the E354A mNAGS and control wild-type NAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was also identified in a patient with hyperammonemia due to NAGS deficiency. Taken together, our results suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
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Effect of arginine on oligomerization and stability of N-Acetylglutamate Synthase.
Scientific reports, 2016Co-Authors: Nantaporn Haskins, Hiroki Morizono, Mendel Tuchman, Amy Mumo, P H Brown, Ljubica CaldovicAbstract:N-Acetylglutamate Synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-Acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-Acetylglutamate Synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS.
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Expression Pattern and Biochemical Properties of Zebrafish N-Acetylglutamate Synthase
PloS one, 2014Co-Authors: Ljubica Caldovic, Mendel Tuchman, Nantaporn Haskins, Amy Mumo, Himani Datta Majumdar, Mary Pinter, Alison KrufkaAbstract:The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-Acetylglutamate Synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate Synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-Acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Kmapp for acetyl coenzyme A increased while the Kmapp for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-Acetylglutamate at the maximal rate.
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A novel biochemically salvageable animal model of hyperammonemia devoid of N-Acetylglutamate Synthase
Molecular genetics and metabolism, 2012Co-Authors: Emilee Senkevitch, Ljubica Caldovic, Hiroki Morizono, Juan Cabrera-luque, Mendel TuchmanAbstract:All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-Acetylglutamate Synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-l-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. Founder NAGS knockout heterozygous mice were obtained from the trans-NIH Knock-Out Mouse Project. Genotyping of the mice was performed by PCR and confirmed by Western blotting of liver and intestine. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags(-/-)) pups and the rescued animals were characterized. We observed an 85% survival rate of Nags(-/-) mice when they were given intraperitoneal injections with NCG and Cit during the newborn period until weaning and supplemented subsequently with both compounds in their drinking water. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 h. In addition to hyperammonemia, interruption of rescue supplementation was associated with elevated plasma glutamine, glutamate, and lysine, and reduced citrulline, arginine, ornithine and proline levels. We conclude that NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.
Hiroki Morizono - One of the best experts on this subject based on the ideXlab platform.
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Gene delivery corrects N-Acetylglutamate Synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-Acetylglutamate Synthase.
Scientific reports, 2021Co-Authors: Parthasarathy Sonaimuthu, Hiroki Morizono, Mendel Tuchman, Nantaporn Haskins, Emilee Senkevitch, Prech Uapinyoying, Markey C. Mcnutt, Ljubica CaldovicAbstract:The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-Acetylglutamate Synthase (NAGS) catalyzes formation of N-Acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
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Impaired ureagenesis due to arginine-insensitive N-Acetylglutamate Synthase
2018Co-Authors: Parthasarathy Sonaimuthu, Hiroki Morizono, Mendel Tuchman, Nantaporn Haskins, Emilee Senkevitch, Prech Uapinyoying, Markey C. Mcnutt, Ljubica CaldovicAbstract:The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to non-toxic urea. N-Acetylglutamate Synthase (NAGS) is an enzyme that catalyzes the formation of N-Acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1), the rate limiting enzyme of the urea cycle. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine but the physiological significance of NAGS activation by L-arginine is unknown. Previously, we have described the creation of a NAGS knockout (Nags-/-) mouse, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG+Cit). In order to investigate the effect of L-arginine on ureagenesis in vivo, we used adeno associated virus (AAV) mediated gene transfer to deliver either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine, to Nags-/- mice. The ability of the E354A mNAGS mutant protein to rescue Nags-/- mice was determined by measuring their activity on the voluntary wheel following NCG+Cit withdrawal. The Nags-/- mice that received E354A mNAGS remained apparently healthy and active but had elevated plasma ammonia concentration despite similar expression levels of the E354A mNAGS and control wild-type NAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was also identified in a patient with hyperammonemia due to NAGS deficiency. Taken together, our results suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
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Effect of arginine on oligomerization and stability of N-Acetylglutamate Synthase.
Scientific reports, 2016Co-Authors: Nantaporn Haskins, Hiroki Morizono, Mendel Tuchman, Amy Mumo, P H Brown, Ljubica CaldovicAbstract:N-Acetylglutamate Synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-Acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-Acetylglutamate Synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS.
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A novel biochemically salvageable animal model of hyperammonemia devoid of N-Acetylglutamate Synthase
Molecular genetics and metabolism, 2012Co-Authors: Emilee Senkevitch, Ljubica Caldovic, Hiroki Morizono, Juan Cabrera-luque, Mendel TuchmanAbstract:All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-Acetylglutamate Synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-l-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. Founder NAGS knockout heterozygous mice were obtained from the trans-NIH Knock-Out Mouse Project. Genotyping of the mice was performed by PCR and confirmed by Western blotting of liver and intestine. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags(-/-)) pups and the rescued animals were characterized. We observed an 85% survival rate of Nags(-/-) mice when they were given intraperitoneal injections with NCG and Cit during the newborn period until weaning and supplemented subsequently with both compounds in their drinking water. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 h. In addition to hyperammonemia, interruption of rescue supplementation was associated with elevated plasma glutamine, glutamate, and lysine, and reduced citrulline, arginine, ornithine and proline levels. We conclude that NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.
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Evolution of the Effect of Arginine on Thermal Stability and Oligomerization of N-Acetylglutamate Synthase
Biophysical Journal, 2011Co-Authors: Ljubica Caldovic, Mendel Tuchman, Nantaporn Haskins, Amy Mumo, Hiroki MorizonoAbstract:N-Acetylglutamate Synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-Acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of arginine biosynthesis pathway, while in animals, NAG acts as an allosteric activator of carbamylphosphate synthetase I and III. NAGS itself is allosterically regulated by arginine. In bacteria, fungi, and plants, arginine acts as an inhibitor, in fish, a partial inhibitor, but in mammals, arginine is an activator. We used Thermofluor methodology to determine if the effect of arginine on the thermal stability of NAGS parallels its effects on NAGS activity. Addition of arginine to bacterial NAGS, which is inhibited by arginine, resulted in a destabilized protein. Addition of arginine to the zebrafish and mouse NAGS stabilized both proteins, despite opposing effects of arginine on their enzymatic activity. We then used analytical gel chromatography to determine if changes in oligomerization state of NAGS could occur upon arginine binding. Our results indicate that bacterial and mammalian NAGS appear to be ensembles of molecules with different oligomerization states that are in rapid exchange with each other. Upon addition of arginine, the partition coefficient of both NAGS increased. The behavior of zebrafish NAGS was different. It eluted as two peaks suggesting two distinct oligomerization states. Upon addition of arginine to zebrafish NAGS the partition coefficients of both peaks decreased. These studies indicate that the effect of arginine on the biophysical properties of NAGS indeed changed during evolution and suggest that the inversion of the allosteric effect and stabilization effects of arginine on NAGS could be linked.
Dashuang Shi - One of the best experts on this subject based on the ideXlab platform.
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Article The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms
2016Co-Authors: Dashuang Shi, Norma M. Allewell, Mendel TuchmanAbstract:Abstract: N-Acetylglutamate Synthase (NAGS) catalyzes the production of N-Acetylglutamate (NAG) from acetyl-CoA and L-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-Acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups
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The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms
International journal of molecular sciences, 2015Co-Authors: Dashuang Shi, Norma M. Allewell, Mendel TuchmanAbstract:N-Acetylglutamate Synthase (NAGS) catalyzes the production of N-Acetylglutamate (NAG) from acetyl-CoA and l-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-Acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups.
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structure of n acetyl l glutamate Synthase kinase from maricaulis maris with the allosteric inhibitor l arginine bound
Biochemical and Biophysical Research Communications, 2013Co-Authors: Mendel Tuchman, Nantaporn Haskins, Dashuang Shi, Zhongmin Jin, Norma M. Allewell, Gengxiang ZhaoAbstract:Abstract Maricaulis maris N -acetylglutamate Synthase/kinase (mmNAGS/K) catalyzes the first two steps in l -arginine biosynthesis and has a high degree of sequence and structural homology to human N -acetylglutamate Synthase, a regulator of the urea cycle. The Synthase activity of both mmNAGS/K and human NAGS are regulated by l -arginine, although l -arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l -arginine, we have determined the structure of the mmNAGS/K complexed with l -arginine at 2.8 A resolution. In contrast to the structure of mmNAGS/K in the absence of l -arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l -arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N -acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l -arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l -arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.
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crystal structure of the n acetyltransferase domain of human n acetyl l glutamate Synthase in complex with n acetyl l glutamate provides insights into its catalytic and regulatory mechanisms
PLOS ONE, 2013Co-Authors: Gengxiang Zhao, Mendel Tuchman, Zhongmin Jin, Norma M. Allewell, Dashuang ShiAbstract:N-Acetylglutamate Synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 A resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-Acetylglutamate Synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.
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Structure of N-acetyl-l-glutamate Synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound.
Biochemical and biophysical research communications, 2013Co-Authors: Gengxiang Zhao, Mendel Tuchman, Nantaporn Haskins, Zhongmin Jin, Norma M. Allewell, Dashuang ShiAbstract:Abstract Maricaulis maris N -acetylglutamate Synthase/kinase (mmNAGS/K) catalyzes the first two steps in l -arginine biosynthesis and has a high degree of sequence and structural homology to human N -acetylglutamate Synthase, a regulator of the urea cycle. The Synthase activity of both mmNAGS/K and human NAGS are regulated by l -arginine, although l -arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l -arginine, we have determined the structure of the mmNAGS/K complexed with l -arginine at 2.8 A resolution. In contrast to the structure of mmNAGS/K in the absence of l -arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l -arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N -acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l -arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l -arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.
M. Dercksen - One of the best experts on this subject based on the ideXlab platform.
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A novel UPLC-MS/MS based method to determine the activity of N-Acetylglutamate Synthase in liver tissue.
Molecular genetics and metabolism, 2016Co-Authors: M. Dercksen, A. Van Cruchten, Mendel Tuchman, Lodewijk Ijlst, Marinus Duran, Wim Kulik, Jos P.n. Ruiter, Ronald J A WandersAbstract:Abstract Background N -acetylglutamate Synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N -acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. Methods UPLC-MS/MS was used in conjunction with stable isotope ( N -acetylglutamic-2,3,3,4,4-d 5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. Results A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N -acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags −/− mice. Conclusion The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
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inhibition of n acetylglutamate Synthase by various monocarboxylic and dicarboxylic short chain coenzyme a esters and the production of alternative glutamate esters
Biochimica et Biophysica Acta, 2014Co-Authors: F. H. Van Der Westhuizen, M. Dercksen, A. Van Cruchten, Lodewyk J Mienie, Lodewijk Ijlst, M Duran, R J A WandersAbstract:Carolina MacGillavry PhD Fellowship awarded by “Koninklijke Nederlandse Akademie van Wetenschappen.”
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Inhibition of N-Acetylglutamate Synthase by various monocarboxylic and dicarboxylic short-chain coenzyme A esters and the production of alternative glutamate esters
Biochimica et Biophysica Acta - Molecular Basis of Disease, 2014Co-Authors: M. Dercksen, A. Van Cruchten, Lodewyk J Mienie, Lodewijk Ijlst, M Duran, F. H. Van Der WesthuizenAbstract:Hyperammonemia is a frequent finding in various organic acidemias. One possible mechanism involves the inhibition of the enzyme N-Acetylglutamate Synthase (NAGS), by short-chain acyl-CoAs which accumulate due to defective catabolism of amino acids and/or fatty acids in the cell. The aim of this study was to investigate the effect of various acyl-CoAs on the activity of NAGS in conjunction with the formation of glutamate esters. NAGS activity was measured in vitro using a sensitive enzyme assay with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) product analysis. Propionyl-CoA and butyryl-CoA proved to be the most powerful inhibitors of N-Acetylglutamate (NAG) formation. Branched-chain amino acid related CoAs (isovaleryl-CoA, 3-methylcrotonyl-CoA, isobutyryl-CoA) showed less pronounced inhibition of NAGS whereas the dicarboxylic short-chain acyl-CoAs (methylmalonyl-CoA, succinyl-CoA, glutaryl-CoA) had the least inhibitory effect. Subsequent work showed that the most powerful inhibitors also proved to be the best substrates in the formation of N-acylglutamates. Furthermore, we identified N-isovalerylglutamate, N-3-methylcrotonylglutamate and N-isobutyrylglutamate (the latter two in trace amounts), in the urines of patients with different organic acidemias. Collectively, these findings explain one of the contributing factors to secondary hyperammonemia, which lead to the reduced in vivo flux through the urea cycle in organic acidemias and result in the inadequate elimination of ammonia.
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Isovaleric acidemia: an integrated approach toward predictive laboratory medicine
2014Co-Authors: M. DercksenAbstract:Isovaleric acidemia (IVA) is an autosomal recessive disorder caused by isovaleryl-CoA dehydrogenase (IVD) deficiency (E.C.1.2.99.10) and patients present with a heterogeneous phenotype, ranging from metabolically mild and/or intermittent to metabolically severe. A cohort of ten IVA patients with a mutual homozygous mutation, c.367G>A (p.G123R), was identified. The IVA patient group however presented with a broad clinical phenotype despite the genetic homogeneity. The homozygous group as well as twelve obligate heterozygotes and relevant control subjects were available for an integrated investigation. This thesis, entitled "Isovaleric acidemia: an integrated approach toward predictive laboratory medicine" presents the outcome of the well-established clinical, biochemical and genetic approach which is followed to characterize an inborn error of metabolism as well as the application of contemporary metabolomics technology consisting of semi-targeted GC-MS analysis as well as bioinformatics applications e.g. PCA, PLS-DA and CONCA, for the disclosure of the metabolic profiles prevailing in the IVA cohort. Furthermore, secondary hyperammonemia observed in IVA was expensively investigated and included the study of N-Acetylglutamate Synthase (NAGS) and the inhibition of this initial step in the urea cycle by isovaleryl-CoA and other short-chain- and short/branched acyl-CoAs. In additional, the study also resulted in the development of novel IVD and NAGS enzyme assays applicable in the diagnostics of IEMs. The integrated outcomes of these approaches opened the possibility to propose a model on future predictive laboratory medicine in the investigations of inherited metabolic diseases.
