Mammalian N-acetylglutamate synthase

Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

doi:10.1016/j.ymgme.2003.10.017

Cited by 40 publications

(33 citation statements)

References 40 publications

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“…The crystals in the absence of L-arginine were hexagonal prisms, whereas those obtained in the presence of L-arginine were hexagonal plates. Activity and Inhibition Assays-NAGS activity measurements were performed using a modification of the assay described by us previously (7,8). The reaction was carried out at 30°C for 1 or 5 min in 100 l of 50 mM Tris-HCl buffer, pH 8.5, containing 100 mM NaCl and 0.2 g of enzyme (unless otherwise specified) and quenched with 100 l of 30% trichloroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

Jin

Caldovic

et al. 2009

Journal of Biological Chemistry

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N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by ϳ10 Å and decreases its height by ϳ20 Å . AAK dimers move 5 Å outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by ϳ4°. The NAT domains rotate ϳ109°relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.L-Arginine biosynthesis in most micro-organisms and plants involves the initial acetylation of L-glutamate by N-acetylglutamate synthase (NAGS, EC 2.3.1.1) 2 to produce N-acetylglutamate (NAG). NAG is then converted by NAG kinase (NAGK, EC 2.7.2.8) to NAG-phosphate and subsequently to N-acetylornithine (1, 2). Two alternative reactions are used to remove the acetyl group from acetylornithine. The linear pathway uses N-acetylornithine deacetylase (EC 3.5.1.16) to catalyze the metal-dependent hydrolysis of the acetyl group to form L-ornithine and acetate, whereas the acetyl recycling pathway transfers the acetyl group from N-acetylornithine to L-glutamate, producing L-ornithine and NAG. This reaction is catalyzed by ornithine acetyltransferase (EC 2.3.1.35).In the linear pathway, NAGS is the only target of feedback inhibition by L-arginine. In contrast, in the acetyl cycling pathway L-arginine may inhibit NAGS and NAGK or ornithine acetyltransferase (3). Structure determinations of L-arginineinsensitive (4) and L-arginine-sensitive NAGKs (5) provided insights into the structural basis of L-arginine inhibition of NAGK. L-Arginine-insensitive Escherichia coli (ec) NAGK is a homodimer (4), whereas L-arginine-sensitive NAGKs from Thermotoga maritima (tm) and Pseudomonas aeruginosa (pa) are hexamers formed by pair-wise interlacing of the N-terminal helices of three ecNAGK-like dimers, to create a second type of dimer interface. L-Arginine binding to a site close to the C terminus induces global conformational changes that expands the ring by ϳ8 Å and decreases the tilt of the ecNAGK-like dimers relative to the plane of the ring by ϳ6°. The inhibition mechanism was proposed to involve the enlargement of an ...

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Section: Methodsmentioning

confidence: 99%

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

Jin

Caldovic

et al. 2009

Journal of Biological Chemistry

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“…A deficiency caused by null alleles results in hyperammonemia early in the newborn period due to the secondary deficiency of CPS-I; without NAG, CPS-I is catalytically inactive [20]. However, late onset hypomorphic alleles also exist, and those patients can often go undiagnosed until adulthood [21,22].…”

Section: Human Disorders and Animal Models N-acetylglutamate Synthasementioning

confidence: 99%

“…Due to its low abundance, a large amount of liver tissue is required in order to perform enzyme activity assays, and this has likely caused many patients to go undiagnosed in the past [24]. However, with the recent cloning and sequencing of the human NAGS gene, a molecular diagnosis of the disease is now possible [20,25].…”

Section: Human Disorders and Animal Models N-acetylglutamate Synthasementioning

confidence: 99%

Contrasting features of urea cycle disorders in human patients and knockout mouse models

Deignan

Cederbaum

Grody

2008

Molecular Genetics and Metabolism

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The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a characteristic profile of plasma amino acid alterations that can be utilized for diagnosis. While enzyme assay is possible, an analysis of the underlying mutation is preferable for an accurate diagnosis. Mouse models for each of the urea cycle disorders exist (with the exception of NAGS deficiency), and for almost all of them, their clinical and biochemical phenotypes rather closely resemble the phenotypes seen in human patients. Consequently, all of the current mouse models are highly useful for future research into novel pharmacological and dietary treatments and gene therapy protocols for the management of urea cycle disorders.

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“…Between each one of these groups, however, the similarities become very weak, suggesting ancient divergence or perhaps even independent origin. Nevertheless the limited similarities found between the DNA segments encoding the C-terminal regions of mammalian and Neurospora NAGS were used to clone the mouse and human NAGS genes (10,51). Moreover, a two-domain structure similar to that of bacterial NAGS was suggested for the mammalian enzyme (51).…”

Section: Glutamate Acetylation In Bacteria N-acetylglutamate Synthasementioning

confidence: 99%

“…Nevertheless the limited similarities found between the DNA segments encoding the C-terminal regions of mammalian and Neurospora NAGS were used to clone the mouse and human NAGS genes (10,51). Moreover, a two-domain structure similar to that of bacterial NAGS was suggested for the mammalian enzyme (51). Of note is that fungal NAGS is active only when associated with NAGK (37, 56), whereas the mammalian gene can complement an E. coli argA auxotroph by itself (10).…”

Section: Glutamate Acetylation In Bacteria N-acetylglutamate Synthasementioning

confidence: 99%

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

Labedan

Glansdorff

2007

Microbiol Mol Biol Rev

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SUMMARY Major aspects of the pathway of de novo arginine biosynthesis via acetylated intermediates in microorganisms must be revised in light of recent enzymatic and genomic investigations. The enzyme N-acetylglutamate synthase (NAGS), which used to be considered responsible for the first committed step of the pathway, is present in a limited number of bacterial phyla only and is absent from Archaea. In many Bacteria, shorter proteins related to the Gcn5-related N-acetyltransferase family appear to acetylate l-glutamate; some are clearly similar to the C-terminal, acetyl-coenzyme A (CoA) binding domain of classical NAGS, while others are more distantly related. Short NAGSs can be single gene products, as in Mycobacterium spp. and Thermus spp., or fused to the enzyme catalyzing the last step of the pathway (argininosuccinase), as in members of the Alteromonas-Vibrio group. How these proteins bind glutamate remains to be determined. In some Bacteria, a bifunctional ornithine acetyltransferase (i.e., using both acetylornithine and acetyl-CoA as donors of the acetyl group) accounts for glutamate acetylation. In many Archaea, the enzyme responsible for glutamate acetylation remains elusive, but possible connections with a novel lysine biosynthetic pathway arose recently from genomic investigations. In some Proteobacteria (notably Xanthomonadaceae) and Bacteroidetes, the carbamoylation step of the pathway appears to involve N-acetylornithine or N-succinylornithine rather than ornithine. The product N-acetylcitrulline is deacetylated by an enzyme that is also involved in the provision of ornithine from acetylornithine; this is an important metabolic function, as ornithine itself can become essential as a source of other metabolites. This review insists on the biochemical and evolutionary implications of these findings.

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Mammalian N-acetylglutamate synthase

Cited by 40 publications

References 40 publications

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

Contrasting features of urea cycle disorders in human patients and knockout mouse models

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

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