Yoshida Takumitsu, Seko Tomohiro, Okada Okimasa, Iwata Kousuke, Liu Lijun, Miki Kunio, Yohda Masafumi
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
Proteins. 2006 Aug 1;64(2):502-12. doi: 10.1002/prot.21010.
X-ray crystallography has revealed two similar alpha/beta domains of the aspartate racemase from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3. The active site is located in the cleft between the two domains where two cysteine residues face each other. This arrangement allows the substrate to enter the cleft and enables the two cysteine residues to act synergistically. However, the distance between their thiolates was estimated to be 9.6 angstroms, which is beyond the distance for cooperative action of them. We examined the molecular mechanism for the racemization reaction of this hyperthermophilic aspartate racemase by mutational analyses and molecular dynamics simulations. The mutational analyses revealed that Arg48 and Lys164 were essential for catalysis in addition to the putative catalytic cysteine residues. The molecular dynamics simulations revealed that the distance between the two active gamma-sulfur atoms of cysteine residues oscillate to periodically become shorter than the predicted cooperative distance at high temperature. In addition, the conformation of Tyr160, which is located at the entrance of the cleft and inhibits the entry of a substrate, changes periodically to open the entrance at 375 K. The opening of the gate is likely to be induced by the motion of the adjacent amino acid, Lys164. The entrance of an aspartate molecule was observed by molecular dynamics (MD) simulations driven by the force of the electrostatic interaction with Arg48, Lys164, and also Asp47. These results provide insights into the roles of amino acid residues at the catalytic site and also the activation mechanism of a hyperthermophilic aspartate racemase at high temperature.
X射线晶体学揭示了嗜热古菌火球菌OT3中天冬氨酸消旋酶的两个相似的α/β结构域。活性位点位于两个结构域之间的裂隙中，两个半胱氨酸残基相对。这种排列方式使底物能够进入裂隙，并使两个半胱氨酸残基协同发挥作用。然而，它们硫醇盐之间的距离估计为9.6埃，这超出了它们协同作用的距离。我们通过突变分析和分子动力学模拟研究了这种嗜热天冬氨酸消旋酶消旋反应的分子机制。突变分析表明，除了假定的催化半胱氨酸残基外，Arg48和Lys164对催化作用也至关重要。分子动力学模拟表明，半胱氨酸残基的两个活性γ-硫原子之间的距离会发生振荡，在高温下会周期性地变得比预测的协同距离短。此外，位于裂隙入口处并抑制底物进入的Tyr160的构象在375 K时会周期性变化以打开入口。入口的打开可能是由相邻氨基酸Lys164的运动诱导的。通过由与Arg48、Lys164以及Asp47的静电相互作用驱动的分子动力学（MD）模拟观察到了天冬氨酸分子的进入。这些结果为催化位点氨基酸残基的作用以及嗜热天冬氨酸消旋酶在高温下的激活机制提供了见解。
