Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

Haskins, Nantaporn; Panglao, Maria; Qu, Qiuhao; Majumdar, Himani Datta; Cabrera-Luque, Juan; Morizono, Hiroki; Tuchman, Mendel; Caldovic, Ljubica

doi:10.1186/1471-2091-9-24

Cited by 34 publications

(77 citation statements)

References 103 publications

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“…280 and Glu 334 , located around the side chain of L-arginine, may also be important for L-arginine binding. The location and orientation of the L-arginine binding in ngNAGS is the same as that identified in tmNAGK (5), and conservation of the L-arginine site between NAGS and NAGK is supported by sitedirected mutagenesis studies of recombinant paNAGS (25), mouse NAGS and bifunctional Xanthomonas campestris (xc) NAGS/K (27). Mutation of key residues binding L-arginine significantly decreased inhibition but had little effect on the kinetic parameters of the protein.…”

Section: Biochemical Characterization Of Ngnags and Inhibition By L-amentioning

confidence: 68%

“…The L-arginine-binding sequence signature, E(I/L)(F/ M)(T/S)XXGXGTX, can be used not only to identify L-arginine-sensitive NAGK (5) but also to identify L-arginine-binding sites in NAGS proteins across phyla. Mutagenesis studies of this region in mouse NAGS and bifunctional xcNAGS/K (27) and paNAGS (25) support the universality of the L-arginine-binding site in NAGS and NAGK. The conservation of this site in mammalian NAGS is noteworthy, because L-arginine activates mammalian NAGS.…”

Section: Conservation Of Nags L-arginine-binding Site Across Phyla-mentioning

confidence: 99%

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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: Biochemical Characterization Of Ngnags and Inhibition By L-amentioning

confidence: 68%

Section: Conservation Of Nags L-arginine-binding Site Across Phyla-mentioning

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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“…3A and Table 1). This effect, which is somewhat reminiscent of the activation of the NAGSs from terrestrial animals by arginine (6,19), suggests that the linker sequence might determine whether arginine is an inhibitor or an activator. The importance not only of the linker length (33) but also of the linker sequence in determining the effect of arginine is illustrated by the observation that the replacement of EAQGP with EAQAF in the modified linker (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…This endows AAK-GNAT domain NAGSs with an enormous potential for adaptation to the specific physiological needs of different organisms. The best example of this ability to adapt is provided by the change in the effect of arginine on NAGS from inhibition to activation with the shift of animals from marine life to terrestrial ureotelism (19), a change that we have partially reproduced by linker manipulation in the present studies.…”

Section: Figmentioning

confidence: 90%

Functional Dissection of N -Acetylglutamate Synthase (ArgA) of Pseudomonas aeruginosa and Restoration of Its Ancestral N -Acetylglutamate Kinase Activity

2012

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In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N -acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased K m glutamate and k cat values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N -acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activity.

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“…Ammonia is toxic to all fish and its effects appear to be similar in fish and mammals. Although fish genomes encode genes for all six urea cycle enzymes and two transporters [29,30], hyperammonemia can be induced in fish simply by immersing them in water with an elevated concentration of ammonia, which is transferred from the water into the blood [31–35]. The enzymatic activity of glutamine synthetase (GS) and the concentration of glutamine are increased in the brains of African sharptooth catfish, rainbow trout and gulf toadfish immersed in water containing sub-lethal concentrations of ammonia [36–38].…”

Section: Introductionmentioning

confidence: 99%

A zebrafish model of hyperammonemia

Feldman¹,

Tuchman²,

Caldovic³

2014

Molecular Genetics and Metabolism

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Hyperammonemia is the principal consequence of urea cycle defects and liver failure, and the exposure of the brain to elevated ammonia concentrations leads to a wide range of neurocognitive deficits, intellectual disabilities, coma and death. Current treatments focus almost exclusively on either reducing ammonia levels through the activation of alternative pathways for ammonia disposal or on liver transplantation. Ammonia is toxic to most fish and its pathophysiology appears to be similar to that in mammals. Since hyperammonemia can be induced in fish simply by immersing them in water with elevated concentration of ammonia, we sought to develop a zebrafish (Danio rerio) model of hyperammonemia. When exposed to 3 mM ammonium acetate (NH4Ac), 50% of 4-day old (dpf) fish died within 3 hours and 4 mM NH4Ac was 100% lethal. We used 4 dpf zebrafish exposed to 4 mM NH4Ac to test whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) and/or NMDA receptor antagonists MK-801, memantine and ketamine, which are known to protect the mammalian brain from hyperammonemia, prolong survival of hyperammonemic fish. MSO, MK-801, memantine and ketamine all prolonged the lives of the ammonia-treated fish. Treatment with the combination of MSO and an NMDA receptor antagonist was more effective than either drug alone. These results suggest that zebrafish can be used to screen for ammonia-neuroprotective agents. If successful, drugs that are discovered in this screen could complement current treatment approaches to improve the outcome of patients with hyperammonemia.

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Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

Cited by 34 publications

References 103 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

Functional Dissection of N -Acetylglutamate Synthase (ArgA) of Pseudomonas aeruginosa and Restoration of Its Ancestral N -Acetylglutamate Kinase Activity

A zebrafish model of hyperammonemia

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