Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
Biochemistry. 2010 Mar 9;49(9):1963-9. doi: 10.1021/bi9015605.
Arylmalonate decarboxylase catalyzes the enantioselective decarboxylation of alpha-aryl-alpha-methylmalonate to produce optically pure alpha-arylpropionate. The enzyme is comprised of two alpha/beta domains and contains an active site situated between the two domains. The site is formed by Tyr48, Gly74-Thr75-Ser76, Tyr126, and Cys188-Gly189-Gly190 residues. Since it has been observed that the Gly74Cys/Cys188Ser mutation inverts the enantioselectivity of the enzyme, we determined the crystal structure of the Gly74Cys/Cys188Ser mutant in the liganded form at a resolution of 1.45 A to understand the structural basis for this inversion. The overall structure of the enzyme overlapped well with that of the benzylphosphonate-associated wild-type enzyme, and the mutations had little effect on the structure of the active site. A ligand molecule bound to the active site in an unusual semiplanar conformation resembling the planar enediolate reaction intermediate could be assigned as phenyl acetate. The inversion in enantioselectivity by the paired mutation is explained by the mirror symmetry between Cys74 in the mutant and Cys188 of the wild type with respect to the carbon atom in the ligand to be protonated. Comparison of the wild-type and Gly74Cys mutant crystal structures suggested that ligand binding induces a positional shift of the Cys188-Gly189-Gly190 region toward the Gly74-Thr75 pair which provides two oxyanion holes necessary to stabilize the negatively charged enediolate reaction intermediate. The ligand binding also simultaneously induces the formation of a hydrophobic cluster over the active site cleft. Thus, AMDase is proposed to have "open" and "closed" conformations of the active site that are regulated by ligand binding. These results may provide an effective strategy for the rational design to invert the enantioselectivity of enzymes.
芳基丙二酸脱羧酶催化α-芳基-α-甲基丙二酸的对映选择性脱羧，生成光学纯的α-芳基丙酸酯。该酶由两个α/β结构域组成，包含位于两个结构域之间的活性位点。该位点由 Tyr48、Gly74-Thr75-Ser76、Tyr126 和 Cys188-Gly189-Gly190 残基形成。由于已经观察到 Gly74Cys/Cys188Ser 突变会使酶的对映选择性反转，因此我们确定了配体形式下 Gly74Cys/Cys188Ser 突变体的晶体结构，分辨率为 1.45Å，以了解这种反转的结构基础。酶的整体结构与苄基膦酸酯相关的野生型酶重叠良好，突变对活性位点的结构影响很小。一个配体分子以不寻常的半平面构象结合到活性位点，类似于平面烯二酮反应中间体，可以被指定为苯乙酸酯。通过配对突变导致的对映选择性反转可以通过突变体中 Cys74 和野生型中 Cys188 相对于要质子化的配体中的碳原子之间的镜像对称性来解释。与野生型和 Gly74Cys 突变体晶体结构的比较表明，配体结合诱导 Cys188-Gly189-Gly190 区域向 Gly74-Thr75 对的位置移动，这提供了稳定带负电荷的烯二酮反应中间体所必需的两个氧阴离子空穴。配体结合还同时诱导活性位点裂缝上形成一个疏水区簇。因此，AMDase 被提议具有受配体结合调节的活性位点的“开放”和“关闭”构象。这些结果可能为合理设计反转酶的对映选择性提供有效的策略。
