Miyashita Yurina, Ohmae Eiji, Nakasone Kaoru, Katayanagi Katsuo
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan.
Extremophiles. 2015 Mar;19(2):479-93. doi: 10.1007/s00792-015-0732-7. Epub 2015 Jan 24.
The effects of salt on the structure, stability, and enzymatic function of a novel dihydrofolate reductase (HjDHFR P1) from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1 living in a Japanese saltern, were studied using ultraviolet absorption, circular dichroism (CD), and fluorescence spectroscopy. HjDHFR P1 had a partial structure at pH 8.0 in the absence of NaCl, and the addition of NaCl (0-500 mM concentration) induced significant structural formation to HjDHFR P1. The addition of NADPH, which is a coenzyme for its catalytic reaction, and lowering the pH from 8 to 6 also induced the same CD change, indicating the formation of the NADPH-binding site in HjDHFR P1. The NaCl dependence of thermal and urea-induced unfolding measurements suggested that protein stability increased depending on NaCl concentration regardless of structural formation, and HjDHFR P1 achieved the same stability as Escherichia coli DHFR at 750 mM NaCl. Halophilic characteristics were also observed for enzymatic function, although its structure had already formed under the conditions that enzymatic activity was measured at due to the presence of NADPH. These results suggest that the halophilic mechanism on structural stability and function was caused by factors other than structural formation, which are suggested to be the contributions of preferential interactions between the protein and salt ions and the specific binding of salt ions.
利用紫外吸收、圆二色性（CD）和荧光光谱法，研究了盐对来自日本盐田嗜盐古菌日本嗜盐碱杆菌TR-1株的新型二氢叶酸还原酶（HjDHFR P1）的结构、稳定性和酶功能的影响。在无NaCl的情况下，HjDHFR P1在pH 8.0时具有部分结构，添加NaCl（浓度为0 - 500 mM）会促使HjDHFR P1形成显著的结构。添加其催化反应的辅酶NADPH以及将pH从8降至6也会诱导相同的CD变化，表明HjDHFR P1中形成了NADPH结合位点。热诱导和尿素诱导的去折叠测量对NaCl的依赖性表明，无论结构形成情况如何，蛋白质稳定性均随NaCl浓度增加而提高，且在750 mM NaCl时，HjDHFR P1达到了与大肠杆菌二氢叶酸还原酶相同的稳定性。尽管在测量酶活性的条件下，由于NADPH的存在其结构已经形成，但在酶功能方面也观察到了嗜盐特性。这些结果表明，结构稳定性和功能上的嗜盐机制是由结构形成以外的因素引起的，推测这些因素是蛋白质与盐离子之间优先相互作用以及盐离子特异性结合的作用。
