The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel

doi:10.1074/jbc.m707678200

Cited by 35 publications

(100 citation statements)

References 51 publications

(62 reference statements)

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“…Active Site and Catalytic Mechanism-L-Glutamate was found to bind to the same site as NAG in our previously reported structure (6). The CoA-and L-glutamate-bound ternary structure further confirms that L-glutamate binds in a site within the NAT domain.…”

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

confidence: 63%

“…However, our recent structural determination of NAGS from Neisseria gonorrhoeae (ng) revealed the active site to be located in the NAT domain, Ͼ25 Å away from the proposed L-arginine-binding site (6). Therefore, the allosteric mechanism of NAGS is likely to be different from that of L-arginine-sensitive NAGKs.…”

mentioning

confidence: 93%

“…Enzyme Production and Crystallization-Cloning, expression, purification, and crystallization of ngNAGS have been previously described (6). Briefly, the protein was expressed in * This work was supported, in whole or in part, by National Institutes of Health Grants DK064913 (to M. T.) and DK067935 (to D. S.).…”

Section: Methodsmentioning

confidence: 99%

“…Analytical Gel Filtration Chromatography-The molecular weight of ngNAGS with or without L-arginine (1.0 mM) was determined with a Superdex 200 HR 10/30 column (Amersham Biosciences) as previously described (6).…”

Section: Methodsmentioning

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-asupporting

confidence: 63%

mentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

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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“…Although the key role and widespread distribution of NAGS in all domains of life warrant studies of this enzyme, most detailed data concern the bacterial forms of this enzyme (17,23,27,32,33), including the crystal structure of NAGS from Neisseria gonorrhoeae (NgNAGS) in the substrate-bound and arginine-bound forms (24,34). NgNAGS can be considered a typical example of classical bacterial NAGSs, as defined by the early-studied Escherichia coli and Pseudomonas aeruginosa enzymes (17,23,32,33).…”

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confidence: 99%

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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Amino Acid Biosynthesis

Parker

Pratt

2010

Amino Acids, Peptides and Proteins in Organic Chemistry

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The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are universally coded by the translation machinery; in addition, two further a-amino acids, selenocysteine and pyrrolysine, are now believed to be incorporated into proteins via ribosomal synthesis in some organisms. More than 300 other amino acid residues have been identified in proteins, but most are of restricted distribution and produced via post-translational modification of the ubiquitous protein amino acids [1]. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination. The most important biosynthetic distinction relates to whether appropriate carbon skeletons are pre-existing in basic metabolism or whether they have to be synthesized de novo and this division underpins the structure of this chapter.There are a small number of a-keto acids ubiquitously found in core metabolism, notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis), together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon skeletons for unbranched a-amino acids of three, four, and five carbons, respectively. a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight chains are made by alternative pathways depending on the available raw materials. The strategic challenge for the biosynthesis of most straight-chain amino acids centers around two issues: how is the a-amino function introduced into the carbon skeleton and what functional group manipulations are required to generate the diversity of side-chain functionality required for the protein function?The core family of straight-chain amino acids does not provide all the functionality required for proteins. a-Amino acids with branched side-chains are used for two purposes; the primary need is related to protein structural issues. Proteins fold into well-defined three-dimensional shapes by virtue of their amphipathic nature: a significant fraction of the amino acid side-chains are of low polarity and the hydrophobic effect drives the formation of ordered structures in which these side-chains are buried away from water. In contrast to the straight-chain amino

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The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

Cited by 35 publications

References 51 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

Amino Acid Biosynthesis

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