Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity
Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity
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DOI：
10.1016/j.jmb.2005.04.029
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发表时间：
2005-06-17
影响因子：
5.6
通讯作者：
Schomburg, D
中科院分区：
文献类型：
--
作者：
Schlieben, NH;Niefind, K;Schomburg, D
短链乳杆菌R-特异醇脱氢酶(RADH)是短链脱氢酶/还原酶(SDR)延伸酶家族中依赖NADP的同源四聚体成员，具有很高的生物技术应用潜力。它在体外的首选底物是前手性酮，如苯乙酮，几乎总是有一个小的甲基作为一个取代基，另一个是大的(通常是芳香的)部分。基于野生型radh与NADP和苯乙酮形成的络合物的原子分辨结构，我们设计了突变体radh-G37D，它应该具有改进的生物技术共底物特异性图谱，即偏好NAD而不是NADP。与野生型和突变体radh的动力学比较表明，这一目标已经实现。为了从结构上鉴定成功的突变体，我们测定了RADH-G37D的部分原子分辨晶体结构，它既是脱辅酶，也是NAD或NADH和苯乙醇的三元络合物。RADH-G37D对NAD(H)亲和力的增强依赖于NAD的腺苷核糖部分与插入的天冬氨酸侧链之间的相互作用。RADH-G37D作为脱辅酶和作为三元复合体的一部分的结构比较表明，Ser141、Glu144、Tyr189和Met205在活性中心附近发生了显著的重排。这种可塑性有助于为RADH底物典型的甲基生成一个小的疏水口袋，并为第二个更可变且通常是芳香族的取代基生成疏水涂层。在Ser141附近，我们甚至在主干中发现了替代构象。我们在这里首次描述了SDR酶在这个区域的结构适应性，这可能在功能上是重要的，因为它与Ser142有关，Ser142是SDR酶典型的高度保守的催化四分体的成员。此外，它还影响一个扩展的质子传递系统，该系统最近被确定为SDR酶催化机制的关键元件。(C)2005爱思唯尔有限公司。保留所有权利。
The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes. (c) 2005 Elsevier Ltd. All rights reserved.