Department of Chemistry, University of Georgia, Athens, Georgia, USA.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
Proteins. 2022 Aug;90(8):1570-1583. doi: 10.1002/prot.26339. Epub 2022 Apr 9.
Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were 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 an R of 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.
三维结构的 I86A 和 C295A 突变的仲醇脱氢酶（SADH）从 Thermoanaerobacter pseudoethanolicus 通过 X 射线晶体学确定。四聚体结构的 C295A-SADH 浸泡与 NADP 和二甲亚砜（DMSO）确定 1.85 ⁇ 与 R 0.225。DMSO 绑定到四面体锌在每个亚基，与配体从 SG 的 Cys-37 ，NE2 的 His-59 ，和 OD2 的 Asp-150。烟酰胺环的 NADP 是氢键的 N 的 Ala-295 和 O 的 Val-265 和 Gly-293。O 的 DMSO 是连接到一个网络的氢键与 OG 的 Ser-39 ，3'-OH 的 NADP ，和 ND1 的 His-42。结构的 I86A-SADH 浸泡与 2-戊醇和 NADP 包含（R）-2-戊醇绑定在每个亚基，连接到四面体锌，和连接到质子传递网络。结构的 I86A-SADH 浸泡与 3-甲基环己醇和 NADP 有酒精绑定在三个亚基。其中两个网站有酒精连接到锌在轴向位置，与 OE2 的 Glu-60 在其他轴向位置的一个三角双锥配合物。一个网站有 3-甲基环己醇绑定非共价键，与锌在一个倒四面体几何与 Glu-60。第四个网站也有锌在一个三角双锥配合物与轴向 Glu-60 和水配体。这些结构表明配体交换的 SADH 涉及五配位和倒锌配合物与 Glu-60。此外，我们看到一个网络的氢键连接的基质氧到外部溶剂，这可能起到了一定的作用在机制的 SADH。
