ANATOMY OF AN ENGINEERED NAD-BINDING SITE
ANATOMY OF AN ENGINEERED NAD-BINDING SITE
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DOI：
10.1002/pro.5560030916
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发表时间：
1994-09-01
期刊：
影响因子：
8
通讯作者：
SCHULZ, GE
中科院分区：
文献类型：
--
作者：
MITTL, PRE;BERRY, A;SCHULZ, GE
大肠杆菌谷胱甘肽还原酶的辅酶特异性通过经由一组点突变：A179 G、A183 G、V197 E、R198 M、K199 F、H200 D和R204 P修饰2 ′-磷酸结合位点的环境而从NADP转换为NAD（Scrutton NS，Berry A，Perham RN，1990，Nature 343：38 - 43）。为了分析所涉及的结构变化，我们确定了4种高分辨率晶体结构，即，野生型酶的结构（1.86度分辨率，R因子为16.8%）、与NADP连接的野生型酶的结构（2.0度，20.8%）、NAD依赖性突变体的结构（1.74度，16.8%）和与NAD连接的NAD依赖性突变体的结构（2.2度，16.9%）。这些结构的比较揭示了细微的差异，解释了特异性变化的细节。特别是，肽旋转发生在腺苷核糖附近，伴随着核糖皱褶的变化。突变导致局部链折叠的收缩。此外，工程化的NAD结合位点呈现比野生型酶的NADP位点刚性更小的结构。连接结构的叠加显示NAD相对于NADP的置换，使得从烟酰胺环到FAD的电子途径延长，这可以解释突变体的较低催化效率。由于烟酰胺距离突变位点多达15埃，这一观察结果提醒我们，突变可能具有难以预测的重要长期后果。
The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 degrees resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 degrees, 20.8%), of the NAD-dependent mutant (1.74 degrees, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 degrees, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 Angstrom from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.