Rizzi M, Nessi C, Mattevi A, Coda A, Bolognesi M, Galizzi A
Department of Genetics and Microbiology A. Buzzati Traverso, Universityof Pavia, Italy.
EMBO J. 1996 Oct 1;15(19):5125-34.
NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three-dimensional structure of NH3-dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with alpha/beta subunit topology. The catalytic site is located at the parallel beta-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed. Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD+ during the first step of the catalyzed reaction. The deamido-NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases.
烟酰胺腺嘌呤二核苷酸合成酶催化烟酰胺腺嘌呤二核苷酸生物合成的最后一步。利用多同晶置换和密度修正技术相结合的方法，通过X射线晶体学（分别在2.6埃和2.0埃分辨率下）解析了来自枯草芽孢杆菌的依赖氨的烟酰胺腺嘌呤二核苷酸合成酶的游离形式及其与ATP复合物的三维结构。该酶由具有α/β亚基拓扑结构的紧密同型二聚体组成。催化位点位于平行β-折叠拓扑转换点，在该位点观察到一个AMP分子、一个焦磷酸和一个Mg2+离子。邻近“P环”的丝氨酸46残基与焦磷酸基团形成氢键，可能在催化反应第一步促进脱酰胺-NAD+的腺苷化过程中发挥作用。位于亚基界面的脱酰胺-NAD+结合位点被一个ATP分子占据，该ATP分子指向催化中心。由丝氨酸46组成的催化位点保守结构指纹与在依赖谷氨酰胺的GMP合成酶中观察到的相关蛋白区域非常相似，支持了烟酰胺腺嘌呤二核苷酸合成酶属于新发现的“N型”ATP焦磷酸酶家族的假说。
