Rizzi M, Bolognesi M, Coda A
Department of Pharmaceutical Science and Technology University of Torino, Italy.
Structure. 1998 Sep 15;6(9):1129-40. doi: 10.1016/s0969-2126(98)00114-2.
Nicotinamide adenine dinucleotide (NAD+) has a central role in life processes. The ubiquitous enzyme NAD+ synthetase catalyzes a key step in NAD+ biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction. NAD+ synthetase belongs to the amidotransferase family and has been recognized as a member of the family of N-type ATP pyrophosphatases. In order to investigate the mechanism of the reaction carried out by NAD+ synthetase we have determined a high-resolution three-dimensional structure of the Bacillus subtilis homodimeric NAD+ synthetase in complex with the trapped reaction intermediate NAD-adenylate.
Two NAD-adenylate molecules and two pyrophosphate (PPi) molecules are observed in the 1.3 A resolution structure of the NAD+ synthetase-NAD-adenylate complex. Structural studies on the NAD+ synthetase-NAD-adenylate adduct and on the cation-binding sites reveal a new deamido-NAD+-binding site located at the subunit interface, locate a binuclear magnesium cluster at the ATP-binding site and, identify two monovalent cation sites, one of which may represent an ammonium-binding site.
Our results suggest that two different catalytic strategies have been adopted by NAD+ synthetase in the two different steps of the reaction. During the adenylation step, no protein residues seem to be located properly to directly participate in catalysis, which is likely to be carried out with the fundamental assistance of an electron-withdrawing trimetallic constellation present in the active site. A different behavior is observed for the second step, in which an ammonium ion is the binding species. In this step, Asp173 is a key residue in both deprotonation of the primarily bound ammonium ion, and stabilization of the tetrahedral transition-state intermediate. Moreover, the structural data suggest that product release can take place only after all substrates are bound to the enzyme, and product release is ultimately controlled by the conformation adopted by two mobile loops.
烟酰胺腺嘌呤二核苷酸（NAD⁺）在生命过程中起核心作用。普遍存在的酶NAD⁺合成酶催化NAD⁺生物合成中的关键步骤，通过两步反应将脱酰胺-NAD⁺转化为NAD⁺。NAD⁺合成酶属于氨基转移酶家族，并且已被认定为N型ATP焦磷酸酶家族的成员。为了研究NAD⁺合成酶所催化反应的机制，我们测定了枯草芽孢杆菌同二聚体NAD⁺合成酶与捕获的反应中间体NAD-腺苷酸复合物的高分辨率三维结构。
在NAD⁺合成酶-NAD-腺苷酸复合物的1.3 Å分辨率结构中观察到两个NAD-腺苷酸分子和两个焦磷酸（PPi）分子。对NAD⁺合成酶-NAD-腺苷酸加合物和阳离子结合位点的结构研究揭示了位于亚基界面的一个新的脱酰胺-NAD⁺结合位点，在ATP结合位点定位了一个双核镁簇，并确定了两个单价阳离子位点，其中一个可能代表铵结合位点。
我们的结果表明，NAD⁺合成酶在反应的两个不同步骤中采用了两种不同的催化策略。在腺苷化步骤中，似乎没有蛋白质残基处于适当位置以直接参与催化，催化可能是在活性位点中存在的吸电子三金属组合的基本协助下进行的。在第二步中观察到不同的行为，其中铵离子是结合物种。在这一步中，Asp173是主要结合的铵离子去质子化以及四面体过渡态中间体稳定化的关键残基。此外，结构数据表明，只有在所有底物都与酶结合后才能发生产物释放，并且产物释放最终由两个可移动环所采用的构象控制。
