Investigating Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods
Investigating Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods
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DOI：
10.1039/c8cy00440d
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发表时间：
2018-10
影响因子：
5
通讯作者：
Robert W. Powell, III;M. Pilar Buteler;Sunidhi Lenka;M. Crotti;Sara Santangelo;Matthew J. Burg;S. Bruner;E. Brenna;A. Roitberg;Jon D. Stewart
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文献类型：
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作者：
Robert W. Powell, III;M. Pilar Buteler;Sunidhi Lenka;M. Crotti;Sara Santangelo;Matthew J. Burg;S. Bruner;E. Brenna;A. Roitberg;Jon D. Stewart
酿酒酵母（Saccharomyces cerevisiae） OYE 3与研究充分的酵母（Saccharomyces pastorianus） OYE 1具有80%的序列一致性；然而，野生型oye3对某些烯烃底物表现出不同的立体选择性。在OYE 3中对Trp 116进行位点饱和诱变，然后进行底物分析，结果表明突变的影响相对较小，与之前在OYE 1中观察到的相反。无配体和酚结合oye3的x射线晶体结构分别被解析到1.8和1.9 A分辨率。这两种结构与oye1几乎相同，仅在活性位点区域有一个氨基酸差异（Ser 296与Phe 296，环6的一部分）。尽管它们的静态x射线结构基本相同，但分子动力学（MD）模拟显示，oye3和oye1在溶液中的环6构象存在显著差异。在OYE 3中，环路6几乎与晶体结构中观察到的一样开放；相比之下，环路6在oye1的活性位点上闭合了约4 A。与OYE 3相比，与OYE 1结合的底物可能产生更多的活性位点蛋白接触。这些差异为oye3和oye1的不同立体选择性提供了解释，尽管它们的x射线晶体结构几乎相同。
Saccharomyces cerevisiae OYE 3 shares 80% sequence identity with the well-studied Saccharomyces pastorianus OYE 1; however, wild-type OYE 3 shows different stereoselectivities toward some alkene substrates. Site-saturation mutagenesis of Trp 116 in OYE 3 followed by substrate profiling showed that the mutations had relatively little effect, opposite to that observed previously for OYE 1. The X-ray crystal structures of unliganded and phenol-bound OYE 3 were solved to 1.8 and 1.9 A resolution, respectively. Both structures were nearly identical to that of OYE 1, with only a single amino acid difference in the active site region (Ser 296 versus Phe 296, part of loop 6). Despite their essentially identical static X-ray structures, molecular dynamics (MD) simulations revealed that loop 6 conformations differed significantly in solution between OYE 3 and OYE 1. In OYE 3, loop 6 remained nearly as open as observed in the crystal structure; by contrast, loop 6 closed over the active site of OYE 1 by ca. 4 A. Loop closure likely generates a greater number of active site protein contacts for substrate bound to OYE 1 as compared to OYE 3. These differences provide an explanation for the differing stereoselectivities of OYE 3 and OYE 1, despite their nearly identical X-ray crystal structures.