Hayashi Junji, Yamamoto Kaori, Yoneda Kazunari, Ohshima Toshihisa, Sakuraba Haruhiko
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0795, Japan.
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, 869-1404, Japan.
Proteins. 2016 Dec;84(12):1786-1796. doi: 10.1002/prot.25161. Epub 2016 Oct 1.
A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V ) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc.
在嗜热古菌嗜热栖热放线菌中鉴定出一种编码sn-甘油-1-磷酸脱氢酶（G1PDH）的基因。该基因在大肠杆菌中过表达，其产物被纯化并进行了表征。与传统的G1PDH不同，表达的酶对NADH表现出强烈的偏好：与NADPH的反应速率（V）仅为与NADH反应速率的2.4%。该酶的晶体结构在2.45 Å的分辨率下确定。不对称单元由一个同型六聚体组成。结构优化和HPLC分析表明，即使在结晶过程中未添加辅因子NADPH，在亚基D、E和F中也存在结合的NADPH。NADPH腺嘌呤核糖C2'处的磷酸基团通过与Ser40和Thr42的五个偏向氢键紧密结合。与已知的G1PDH结构相比，观察到NADPH分子被从正常的辅酶结合位点推开。有趣的是，S40A/T42A双突变酶与NADPH的反应活性比野生型酶高得多，这表明C2'-磷酸基团周围的偏向相互作用使NADPH结合不足以进行催化。我们的结果为NAD(P)依赖性脱氢酶中辅酶偏好提供了独特的结构基础。《蛋白质》2016年；84:1786 - 1796。© 2016威利期刊公司。
