N-acetylglutamate synthase (NAGS) is an enzyme that produces the cognate cofactor N-acetylglutamate (NAG), an essential allosteric activator of the first and rate limiting enzyme of ureagenesis (CPS I) in mammals, and the first committed substrate for arginine biosynthesis in microorganisms. Our cloning and expression of the mouse and human NAGS genes and many other NAGS genes from various species, makes it now possible to gain structure/function insights into this interesting protein. We found in a number of proteobacteria species (X. campestris, M. maris, O. alexandrii, X. axonopodis, and X. fastidiosa Dixon) a gene for a bifunctional NAGS/NAGK that is similar to mammalian NAGS and for which we have obtained protein crystals. Recently, we obtained a high quality density map which will lead to the determination of the first three-dimensional structure of NAGS (from N. gonorrhoeae). Since NAGS is a likely regulator of ureagenesis and its function is allosterically affected by arginine, it is now possible to understand the mechanism(s) of the arginine effect and to compare the regulation of NAGS in hepatic vs. intestinal, tissues. The deficiency of NAG in inherited NAGS deficiency, organic acidemias and valproate treatment causes hyperammonemia that frequently leads to brain damage, developmental disabilities and death. Better understanding of the NAG/NAGS system will improve the diagnosis and treatment of these conditions.
The specific aims of this project are 1) To solve the liganded and unliganded structures of NAGS and characterize mechanisms for catalysis and the effect of arginine;2) To characterize the biochemical properties of NAGS proteins across phyla, focusing on the effect of arginine on structure and function;3) To differentiate regulatory mechanisms that are specific to liver ureagenesis by characterizing and comparing the regulation of NAGS expression in liver and intestine;4) To determine the functional effects of naturally-occurring mutations that cause inherited NAGS deficiency. Biochemical, crystallographic and molecular methods will be employed to gain an in depth understanding of the structural biology, biochemistry, pathophysiology, genotype/phenotype correlations of the NAGS genes and proteins in the context of evolutionary development of this system. After the first three- dimensional structure of NAGS has been solved, other structures of refractory NAGS proteins will become available. This will lead to constructing a structural model of mammalian NAGS, deriving at a catalytic mechanism, determining the mechanism of arginine effect, and the effects of mutations causing NAGS dysfunction and hyperammonemia.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Special Emphasis Panel (ZRG1-GTIE-A (01))
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Mckeon, Catherine T
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Children's Research Institute
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Williams, Monique; Burlina, Alberto; Rubert, Laura et al. (2018) N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-acetylglutamate Synthase Enhancer Region. Sci Rep 8:15436
Shi, Dashuang; Zhao, Gengxiang; Ah Mew, Nicholas et al. (2017) Precision medicine in rare disease: Mechanisms of disparate effects of N-carbamyl-l-glutamate on mutant CPS1 enzymes. Mol Genet Metab 120:198-206
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Shi, Dashuang; Allewell, Norma M; Tuchman, Mendel (2015) From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases. Int J Mol Sci 16:18836-64
Shi, Dashuang; Allewell, Norma M; Tuchman, Mendel (2015) The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms. Int J Mol Sci 16:13004-22
Ah Mew, Nicholas; McCarter, Robert; Daikhin, Yevgeny et al. (2014) Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1 deficiency with N-carbamyl-L-glutamate. J Pediatr 165:401-403.e3
Caldovic, Ljubica; Haskins, Nantaporn; Mumo, Amy et al. (2014) Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase. PLoS One 9:e85597
Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M et al. (2013) 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 8:e70369
Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin et al. (2013) Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound. Biochem Biophys Res Commun 437:585-90

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