Structural Insights into Phosphite Dehydrogenase Variants Favoring a Non-natural Redox Cofactor
Structural Insights into Phosphite Dehydrogenase Variants Favoring a Non-natural Redox Cofactor
复制标题
对有利于非天然氧化还原辅因子的亚磷酸脱氢酶变体的结构见解
DOI：
10.1021/acscatal.8b04822
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发表时间：
2019-03-01
期刊：
影响因子：
12.9
通讯作者：
Zhao, Zongbao Kent
中科院分区：
文献类型：
--
作者：
Liu, Yuxue;Feng, Yanbin;Zhao, Zongbao Kent
实现普遍存在的氧化还原辅因子烟酰胺腺苷二核苷酸（NAD）的非天然辅因子替代物具有极大的科学和生物技术兴趣。几种氧化还原酶已经被改造成有利于烟酰胺胞嘧啶二核苷酸（NCD），一种较小尺寸的NAD类似物。然而，涉及NAD类似物的分子相互作用仍然难以捉摸，阻止我们设计更多的酶来接受这些类似物。在这里，我们采取了一种半理性的方法来发展亚磷酸脱氢酶（Pdh），并确定了具有大幅改善的NCD偏好的变体。这些突变体是利用亚磷酸盐作为电子供体再生还原NCD的有价值的组分。然后，我们收集了三种Pdh变体及其NCD复合物的X射线晶体结构，以描绘NCD结合的分子基础。发现具有包围NAD结合口袋的大侧链的氨基酸残基的掺入导致有利于NCD超过NAD的紧凑环境，以及NCD与这些侧链之间的额外相互作用。这些结果指导了具有良好NCD偏好的更多Pdh突变体的成功工程化。由于许多氧化还原酶具有关键的结构特征，因此我们的策略可以很容易地用于设计有利于NCD的酶。我们预计，在不久的将来，将创建更多与非天然辅因子相关的合成系统，作为广泛应用的替代工具，以解决化学和合成生物学家面临的挑战性问题。
Implementation of a non-natural cofactor alternative to the ubiquitous redox cofactor nicotinamide adenosine dinucleotide (NAD) is of great scientific and biotechnological interest. Several redox enzymes have been engineered to favor nicotinamide cytosine dinucleotide (NCD), a smaller-sized NAD analogue. However, molecular interactions involving NAD analogues remain elusive, preventing us from devising more enzymes to accept those analogues. Here we took a semirational approach to evolve phosphite dehydrogenase (Pdh) and identified variants with substantially improved NCD preference. These mutants are valuable components for regeneration of reduced NCD by using phosphite as the electron donor. We then collected X-ray crystal structures of three Pdh variants and their NCD-complexes to delineate molecular basis for NCD binding. It was found that the incorporation of amino acid residues with large side chains enclosing the NAD-binding pocket led to compacted environment favoring NCD over NAD, and additional interactions between NCD and these side chains. These results guided successful engineering of more Pdh mutants with good NCD preference. As many redox enzymes share key structural features, our strategy may be readily adopted to devise NCD-favoring enzymes. We expected that, in the near future, more synthetic systems linked to non-natural cofactors will be created as alternative tools for widespread applications to address challenging problems by chemical and synthetic biologists.