Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.
Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.
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来自拟乙醇嗜热厌氧杆菌的嗜热仲醇脱氢酶三元复合物的晶体快照揭示了配体交换和质子中继网络的动态。
DOI：
10.1002/prot.26339
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发表时间：
2022
期刊：
影响因子：
2.9
通讯作者：
Phillips,RobertS
中科院分区：
文献类型：
--
作者：
Dinh,Tung;Rahn,KTroy;Phillips,RobertS
用X射线晶体学方法测定了拟乙醇热厌氧杆菌（Thermoanaerodynamicpseudoethanolicus）中I86 A和C295 A突变型仲醇脱氢酶（SADH）的三维结构。用NADP+和二甲亚砜（DMSO）浸泡的C295 A-SADH的四聚体结构测定为1.85 μ g，其中Rfreeof为0.225。DMSO与每个亚基中的四面体锌结合，配体来自Cys-37的SG、His-59的NE 2和Asp-150的OD 2。NADP的烟酰胺环与Ala-295的N以及瓦尔-265和Gly-293的O形成氢键。DMSO的O与Ser-39的OG、NADP的3′-OH和His-42的ND 1连接到氢键网络。用2-戊醇和NADP+浸泡的I86 A-SADH的结构包含结合在每个亚基中的（R）-2-戊醇，连接到四面体锌，并连接到质子中继网络。用3-甲基环己醇和NADP+浸泡的I86 A-SADH的结构具有结合在三个亚基中的醇。其中两个位点在轴向位置上将乙醇连接到锌上，而Glu-60的OE 2位于三角双锥复合物的另一个轴向位置上。一个位点具有非共价键合的3-甲基环己醇，其中锌与Glu-60呈倒四面体几何结构。第四个位点也有一个三角双锥配合物中的锌与轴向Glu-60和水配体。这些结构表明，SADH的配体交换涉及与Glu-60的五配位和反向锌络合物。此外，我们看到一个网络的氢键连接的底物氧的外部溶剂，这是可能发挥作用的机制SADH。
Three‐dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) fromThermoanaerobacter pseudoethanolicuswere determined by x‐ray crystallography. The tetrameric structure of C295A‐SADH soaked with NADP+and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with anRfreeof 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys‐37, NE2 of His‐59, and OD2 of Asp‐150. The nicotinamide ring of NADP is hydrogen‐bonded to the N of Ala‐295 and the O of Val‐265 and Gly‐293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser‐39, the 3′‐OH of NADP, and ND1 of His‐42. The structure of I86A‐SADH soaked with 2‐pentanol and NADP+contains (R)‐2‐pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A‐SADH soaked with 3‐methylcyclohexanol and NADP+has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu‐60 in the other axial position of a trigonal bipyramidal complex. One site has 3‐methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu‐60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu‐60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu‐60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.