Symersky Jindrich, Devedjiev Yancho, Moore Karen, Brouillette Christie, DeLucas Larry
Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 35294, USA.
Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1138-46. doi: 10.1107/s0907444902006698. Epub 2002 Jun 20.
The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase. This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5. The crystal was exposed to ammonium ions, synchrotron diffraction data were collected and the atomic model was refined anisotropically at 1 A resolution to R = 11.63%. Both binding sites are occupied by the NAD-adenylate intermediate, pyrophosphate and two magnesium ions. The atomic resolution of the structure allows better definition of non-planar peptide groups, reveals a low mean anisotropy of protein and substrate atoms and indicates the H-atom positions of the phosphoester group of the reaction intermediate. The phosphoester group is protonated at the carbonyl O atom O7N, suggesting a carbenium-ion structure stabilized by interactions with two solvent sites presumably occupied by ammonia and a water molecule. A mechanism is proposed for the second catalytic step, which includes a nucleophilic attack by the ammonia molecule on the intermediate.
NAD⁺生物合成的最后一步包括由NAD⁺合成酶催化将酰胺转移至烟酸腺嘌呤二核苷酸（NaAD）。该酶在微重力条件下于pH 8.5时与天然底物NaAD和ATP共结晶。晶体暴露于铵离子中，收集同步加速器衍射数据，并在1 Å分辨率下对原子模型进行各向异性精修，R值为11.63%。两个结合位点均被NAD - 腺苷酸中间体、焦磷酸和两个镁离子占据。该结构的原子分辨率有助于更好地定义非平面肽基团，揭示蛋白质和底物原子的低平均各向异性，并表明反应中间体磷酸酯基团的氢原子位置。磷酸酯基团在羰基O原子O7N处质子化，表明通过与可能被氨和水分子占据的两个溶剂位点相互作用而稳定的碳正离子结构。提出了第二步催化反应的机制，其中包括氨分子对中间体的亲核攻击。
