Cherney Leonid T, Cherney Maia M, Garen Craig R, Niu Chunying, Moradian Fatemeh, James Michael N G
Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
J Mol Biol. 2007 Apr 13;367(5):1357-69. doi: 10.1016/j.jmb.2007.01.033. Epub 2007 Jan 20.
The enzyme N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductive dephosphorylation of N-acetyl-gamma-glutamyl-phosphate to N-acetylglutamate-gamma-semialdehyde. This reaction is part of the arginine biosynthetic pathway that is essential for some microorganisms and plants, in particular, for Mycobacterium tuberculosis (Mtb). The structures of apo MtbAGPR in the space groups P2(1)2(1)2(1) and C2 and the structure of MtbAGPR bound to the cofactor NADP(+) have been solved and analyzed. Each MtbAGPR subunit consists of alpha/beta and alpha+beta domains; NADP(+) is bound in the cleft between them. The hydrogen bonds and hydrophobic contacts between the enzyme and cofactor have been examined. Comparison of the apo and the bound enzyme structures has revealed a conformational change in MtbAGPR upon NADP(+) binding. Namely, a loop (Leu88 to His92) moves more than 5 A to confine sterically the cofactor's adenine moiety in a hydrophobic pocket. To identify the catalytically important residues in MtbAGPR, a docking of the substrate to the enzyme has been performed using the present structure of the MtbAGPR/NADP(+) complex. It reveals that residues His217 and His219 could form hydrogen bonds with the docked substrate. In addition, an ion pair could form between the substrate phosphate group and the guanidinium group of Arg114. These interactions optimally place and orient the substrate for subsequent nucleophilic attack by Cys158 on the substrate gamma-carboxyl group. His219 is the most probable general base to accept a proton from Cys158 and an adjacent ion pair interaction with the side-chain carboxyl group of Glu222 could help to stabilize the resulting positive charge on His219. For this catalytic triad to function efficiently it requires a small conformational change of the order of 1 A in the loop containing His217 and His219; this could easily result from the substrate binding.
N - 乙酰 - γ - 谷氨酰磷酸还原酶（AGPR）催化烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖的N - 乙酰 - γ - 谷氨酰磷酸还原脱磷酸反应，生成N - 乙酰谷氨酸 - γ - 半醛。该反应是精氨酸生物合成途径的一部分，对某些微生物和植物至关重要，特别是对结核分枝杆菌（Mtb）。已解析并分析了空间群为P2(1)2(1)2(1)和C2的无辅基MtbAGPR的结构以及与辅因子NADP(+)结合的MtbAGPR的结构。每个MtbAGPR亚基由α/β结构域和α + β结构域组成；NADP(+)结合在它们之间的裂隙中。已研究了酶与辅因子之间的氢键和疏水相互作用。无辅基酶结构与结合辅因子的酶结构的比较揭示了MtbAGPR在结合NADP(+)后发生的构象变化。具体而言，一个环（Leu88至His92）移动超过5埃，在空间上限制辅因子的腺嘌呤部分位于一个疏水口袋中。为了确定MtbAGPR中具有催化重要性的残基，利用MtbAGPR/NADP(+)复合物的当前结构对底物与酶进行了对接。结果表明，His217和His219残基可与对接的底物形成氢键。此外，底物磷酸基团与Arg114的胍基之间可形成离子对。这些相互作用以最佳方式定位并定向底物，以便随后Cys158对底物γ - 羧基进行亲核攻击。His219最有可能作为从Cys158接受质子的通用碱，并且与Glu222侧链羧基的相邻离子对相互作用有助于稳定His219上产生的正电荷。为使这个催化三联体有效发挥作用，它需要在包含His217和His219的环中发生约1埃的小构象变化；这很容易由底物结合引起。
