Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Korea.
Mol Cells. 2011 Sep;32(3):257-64. doi: 10.1007/s10059-011-1047-x. Epub 2011 Jul 15.
A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys(112) between the active Cys(51) and Cys(171) residues. In the present study, additional Cys residues (Cys(31), Cys(112), and Cys(192)) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments.
先前已经对一种在典型的 2-半胱氨酸过氧化物酶（2-Cys Prx）样蛋白（PpPrx）中起作用的蛋白进行了研究，这种蛋白在假单胞菌 KT2440 中可以作为过氧化物酶或分子伴侣。PpPrx 的双重功能是由在活性半胱氨酸（Cys51）和半胱氨酸（Cys171）残基之间存在的另外一个半胱氨酸（Cys112）来调节的。在本研究中，向 PpPrx 变体中添加了额外的半胱氨酸残基（Cys31、Cys112 和 Cys192）以改善其酶功能。评估了额外半胱氨酸残基在双重功能中的最佳位置。与野生型相比，S31C 和 Y192C 突变体的过氧化物酶活性提高了 3 到 4 倍，而伴侣活性保持在 PpPrx 的>66%。为了研究双重功能的优化是否可以增强体内的应激耐受性，进行了互补研究。在热和氧化应激的复杂条件下，S31C 和 Y192C 突变体比其他变体具有更高的耐受性。优化后的 PpPrx 双重功能可以适应于生物工程系统和工业，例如开发对极端环境更具抗性的生物。
