Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases
Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases
复制标题
DOI：
10.1073/pnas.0601605103
复制
发表时间：
2006-06-27
影响因子：
11.1
通讯作者：
Chappell, Joe
中科院分区：
文献类型：
--
作者：
Greenhagen, Bryan T.;O'Maille, Paul E.;Chappell, Joe
萜烯脱氢酶是一个机制上有趣的酶家族，其催化复杂的多步反应，能够产生数百种具有生物学和商业重要性的结构多样的烃和含氧支架。有趣的是，从真菌到植物的远亲萜烯脱氢酶都含有不变的三维折叠，并且它们的活性位点的分子比较表明它们富含不与反应中间体直接反应的相对惰性的氨基酸残基。因此，催化特异性似乎依赖于活性位点的轮廓和动力学，所述活性位点由氨基酸主链和侧链在该催化表面上的定位以及由围绕合成酶活性位点腔的残基的支持层产生。尽管萜烯脱氢酶之间的高度结构相关性，以前的研究表明，没有明确的关系之间的组织和催化特异性是很容易破译。我们现在基于活性位点内和周围可变残基的系统识别和突变替换，报告了两种不同但进化相关的萜合酶之间催化特异性的相互转化。此外，我们发现以前没有记录的生物合成活动的相互转换过程中，活动可能已经存在于一个共同的祖先，这两个高度相关的酶。这些结果提供了一个简化的手段映射的结构特征，负责功能属性和一种策略，用于识别残基，区分不同的生物合成特性在遗传相关的萜烯脱氢酶。
Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.