Aihara Takayuki, Ito Toshiya, Yamanaka Yasuaki, Noguchi Keiichi, Odaka Masafumi, Sekine Masae, Homma Hiroshi, Yohda Masafumi
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan.
Extremophiles. 2016 Jul;20(4):385-93. doi: 10.1007/s00792-016-0829-7. Epub 2016 Apr 19.
Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.
对嗜酸嗜热古菌嗜热栖热放线菌的5'-磷酸吡哆醛非依赖性天冬氨酸消旋酶进行了功能和结构表征。嗜热栖热放线菌天冬氨酸消旋酶对天冬氨酸表现出高底物特异性。最佳反应温度为60°C，几乎与最佳生长温度相同。反映出细胞质中的低pH值，嗜热栖热放线菌天冬氨酸消旋酶的最佳反应pH值约为5.5。然而，在假定的细胞质pH值4.6下的活性比在最佳pH值5.5下的活性低约6倍。嗜热栖热放线菌天冬氨酸消旋酶的晶体结构与其他5'-磷酸吡哆醛非依赖性天冬氨酸消旋酶的晶体结构几乎相同。在二聚体的两个分子中，一个分子在催化位点含有一个酒石酸分子；另一个分子的结构相对灵活。最后，我们检测了细胞内D-氨基酸的存在情况。出乎意料的是，D-天冬氨酸占总天冬氨酸的比例不是很高。相比之下，观察到了D-脯氨酸和D-丙氨酸。由于嗜热栖热放线菌天冬氨酸消旋酶对天冬氨酸具有高度特异性，嗜热栖热放线菌中可能存在其他氨基酸消旋酶。
