Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering
Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering
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DOI：
10.1074/jbc.m304147200
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发表时间：
2003-08-08
影响因子：
4.8
通讯作者：
Vanoni, MA
中科院分区：
文献类型：
--
作者：
Petoukhov, MV;Svergun, DI;Vanoni, MA
巴西固氮菌谷氨酸合酶（Azoacellum brasilense glutamate synthase，GltS）是细菌NADPH依赖性酶的原型，是一类在氨同化过程中必不可少的复杂铁硫黄素蛋白。使用同步辐射X射线溶液散射分析了催化活性GltS alphaeta全酶及其分离的α和β亚基（分别为162和52 kDa）。发现GltS α亚基和alphabeta全酶在溶液中是四聚体，而β亚基是单体和二聚体的混合物。从散射数据恢复的从头算低分辨率形状表明，（alphabeta）（4）全酶中的亚基的排列与四聚体α（4）复合物中的亚基的排列相似，β亚基占据全酶的外围。假设P222对称性，使用单体α亚基的可用晶体学坐标进一步建模α（4）的结构。为了模拟整个alphabeta全酶，一个假定的alphabeta原聚体构建的α亚基和猪二氢嘧啶脱氢酶，这是类似于β亚基的N-末端区域的坐标。刚体精修产生了GltS模型，其α亚基的排列与α中的相似（4），但也显示了属于相邻原聚体的β亚基之间的接触。全酶模型允许独立的催化活性的alphaeta原聚体，这是与现有的生化证据。
Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alphabeta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alphabeta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of subunits in the (alphabeta)(4) holoenzyme is similar to that in the tetrameric alpha(4) complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha(4) was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alphabeta holoenzyme, a putative alphabeta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha(4), but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alphabeta protomers, which is consistent with the available biochemical evidence.