Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis
Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis
复制标题
Pyrobaculum calidifontis 超嗜热古菌 sn-甘油-1-磷酸脱氢酶的独特辅酶结合模式
DOI：
10.1002/prot.25161
复制
发表时间：
2016
期刊：
影响因子：
2.9
通讯作者：
Haruhiko Sakuraba
中科院分区：
文献类型：
--
作者：
Junji Hayashi;Kaori Yamamoto;Kazunari Yoneda;Toshihisa Ohshima;Haruhiko Sakuraba
在嗜热嗜热古细菌中发现了编码甘油-1-磷酸脱氢酶(G1PDH)的基因。在大肠杆菌中高效表达了该基因，并对其产物进行了纯化和鉴定。与传统的G1PDH相比，表达的酶对NADH表现出强烈的选择性：与NADPH的反应速度(Vmax)仅为与NADH的反应速度的2.4%。测定了该酶的晶体结构，其分辨率为2.45？不对称单位由一个同源六聚体组成。结构的改进和高效液相分析表明，即使在结晶过程中没有添加，D、E和F亚基中也存在结合的辅因子NADPH。NADPH的腺嘌呤核糖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分子被推离了正常的辅酶结合部位。有趣的是，S40A/T42A双突变酶与NADPH的反应活性远高于野生型酶，这表明C2‘-磷酸基团周围的偏向相互作用使NADPH结合不足以催化。我们的结果为依赖NAD(P)的脱氢酶的辅酶偏好提供了独特的结构基础。蛋白质2016；84：1786-1796。©2016 Wiley期刊，Inc.
A gene encoding ansn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeonPyrobaculum calidifontis. The gene was overexpressed inEscherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (Vmax) 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.