HIGH RESOLUTION STRUCTURES OF HOLO AND APO FORMATE DEHYDROGENASE
HIGH RESOLUTION STRUCTURES OF HOLO AND APO FORMATE DEHYDROGENASE
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DOI：
10.1006/jmbi.1994.1188
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发表时间：
1994-02-25
影响因子：
5.6
通讯作者：
WILSON, KS
中科院分区：
文献类型：
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作者：
LAMZIN, VS;DAUTER, Z;WILSON, KS
来自甲基营养细菌Pseudomonassp的holo（三元复合酶-NAD-叠氮化物）和apo NAD依赖的二聚甲酸脱氢酶（FDH）的三维晶体结构。在2.05和1.80 μ m分辨率下，101的R因子分别为11.7%和14.8%。原子坐标的均方根误差估计为0.11 π/holo和0.18 π/apo。使用成像板扫描仪和同步辐射从单晶中收集X射线数据。在两种晶体形式中，在不对称单元中存在二聚体。这两种结构基本上都显示出2重分子对称性。NAD结合导致催化结构域的移动和C末端的有序化，在那里出现新的螺旋。这完成了全FDH中酶活性中心的形成。NAD结合在分隔结构域的裂缝中，并且主要与来自辅酶结合结构域的残基相互作用。在apo FDH中，这些残基通过占据NAD结合区的水分子保持基本相同的构象。叠氮化物分子位于催化点，NAD的烟酰胺部分的C4原子，并与提出的甲酸盐结合重叠。有一个广泛的通道从活性位点运行到蛋白质表面，这应该是由底物使用，以达到活性中心后，NAD已经结合。活性中心的结构和一个假设的催化机理进行了讨论。根据FDH的三维结构，讨论了FDH与其它NAD依赖性甲酸酯酶和某些特定的甲酸酯酶的序列同源性。
Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacteriumPseudomonassp. 101 have been refined toRfactors of 11·7% and 14·8% at 2·05 and 1·80 Å resolution, respectively. The estimated root-mean-square error in atomic co-ordinates is 0·11 Å for holo and 0·18 Å for apo. X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation. In both crystal forms there is a dimer in the asymmetric unit. Both structures show essentially 2-fold molecular symmetry.NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears. This completes formation of the enzyme active centre in holo FDH. NAD is bound in the cleft separating the domains and mainly interacts with residues from the co-enzyme binding domain. In apo FDH these residues are held in essentially the same conformation by water molecules occupying the NAD binding region. An azide molecules is located the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding. There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active centre after NAD has already bound. The structure of the active site and a hypothetical catalytic mechanism are discussed. Sequence homology of FDH with other NAD-dependent formate dehydrogenases and somed-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure.