Department of Life Science and Biotechnology, Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu 564-8680, Japan.
Chem Biodivers. 2010 Jun;7(6):1591-602. doi: 10.1002/cbdv.200900258.
We found that a single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site-directed mutagenesis or reduced with dithiothreitol (DTT). The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The substrate specificity and the overall structure did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before protein maturation. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity.
我们发现，假单胞菌属 NBRC 3460 的精氨酸消旋酶（ArgR）的一级结构中存在半胱氨酸 C47 和 C73 之间的单个分子内二硫键，这是第一个含有二硫键的吡哆醛磷酸（PLP）依赖性氨基酸消旋酶的例子。当 ArgR 的二硫键被定点突变或用二硫苏糖醇（DTT）还原时，仍然检测到氨基酸消旋酶活性。当 ArgR 的二硫键被定点突变破坏时，ArgR 的热和 pH 曲线以及四级结构没有改变。当 ArgR 的二硫键在蛋白质成熟后用 DTT 还原时，ArgR 的底物特异性和整体结构没有改变。然而，当 ArgR 的二硫键在蛋白质成熟之前被定点突变破坏时，这些特性发生了变化。当 ArgR 的二硫键在蛋白质成熟之前被定点突变破坏或 ArgR 在细胞质中表达时，ArgR 的总活性降低。基于这些结果，我们可以得出结论，ArgR 的二硫键对于 ArgR 作为具有广泛底物特异性的氨基酸消旋酶折叠和成熟是必不可少的。
