Center for Genetic Medicine Research and Department of Integrative Systems Biology, Children's National Medical Center, The George Washington University, Washington, DC, United States of America.
PLoS One. 2013 Jul 24;8(7):e70369. doi: 10.1371/journal.pone.0070369. Print 2013.
N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.
N-乙酰谷氨酸合酶（NAGS）催化乙酰辅酶 A 和 L-谷氨酸转化为辅酶 A 和 N-乙酰-L-谷氨酸（NAG），NAG 是尿素循环中氨甲酰磷酸合成酶 I（CPSI）的必需辅因子。NAGS 缺乏会导致血液中氨水平升高，从而产生神经毒性。我们在此报告了人类 NAGS 的首个晶体结构，即与 N-乙酰-L-谷氨酸结合的催化 N-乙酰转移酶（hNAT）结构域，分辨率为 2.1Å。功能研究表明，hNAT 结构域在没有氨基酸激酶（AAK）结构域的情况下保留催化活性。相反，AAK 结构域的主要功能似乎是为别构激活剂 L-精氨酸提供结合位点，以及一个可能在信号转导到 CPS1 中起作用的 N 端脯氨酸丰富基序。结晶 hNAT 形成二聚体，类似于来自 Maricaulis maris 的双功能 N-乙酰谷氨酸合酶/激酶（NAGS/K）中形成的 NAT-NAT 二聚体，并且在溶液中也以二聚体形式存在。NAG 结合位点的结构与突变研究一起，提供了对催化机制的深入了解。我们还表明，来自人类和小鼠的天然 NAGS 以类似于双功能 NAGS/K 的四聚体形式存在。
