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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK064913-09
Application #
8248179
Study Section
Special Emphasis Panel (ZRG1-GTIE-A (01))
Program Officer
Mckeon, Catherine T
Project Start
2003-08-15
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
9
Fiscal Year
2012
Total Cost
$328,065
Indirect Cost
$124,590
Name
Children's Research Institute
Department
Type
DUNS #
143983562
City
Washington
State
DC
Country
United States
Zip Code
20010
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; Allewell, Norma M; Tuchman, Mendel et al. (2013) Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog. Biochem Biophys Res Commun 430:1253-8
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
Heibel, Sandra Kirsch; Lopez, Giselle Yvette; Panglao, Maria et al. (2012) Transcriptional regulation of N-acetylglutamate synthase. PLoS One 7:e29527
Senkevitch, Emilee; Cabrera-Luque, Juan; Morizono, Hiroki et al. (2012) A novel biochemically salvageable animal model of hyperammonemia devoid of N-acetylglutamate synthase. Mol Genet Metab 106:160-8
Heibel, Sandra K; Ah Mew, Nicholas; Caldovic, Ljubica et al. (2011) N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy. Hum Mutat 32:1153-60
Nissim, Itzhak; Horyn, Oksana; Nissim, Ilana et al. (2011) Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase. J Biol Chem 286:22055-68
Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan et al. (2011) A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris. PLoS One 6:e28825

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