Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, Washington 99164-6340, United States.
Biochemistry. 2020 May 5;59(17):1661-1664. doi: 10.1021/acs.biochem.0c00206. Epub 2020 Apr 21.
Monoterpene synthases catalyze the first committed step in the biosynthesis of monoterpenes and are in part responsible for the enormous structural diversity among this class of metabolites. Here, we explore the structure-function relationships underlying the formation of limonene enantiomers in limonene synthases that bind geranyl diphosphate as a common substrate. On the basis of analyses that consider both crystal structure data and amino acid sequence divergence, we identified candidate active site residues with potential roles in catalyzing reactions that involve accommodating reaction intermediates of opposite enantiomeric series. We demonstrate that spearmint (-)-limonene synthase [which generates >99% (-)-limonene over (+)-limonene] can be converted into a mutant enzyme, by exchanging four residues (C321S, N345I, I453V, and M458V), which produces (+)-limonene with reversed enantiospecificity [80% (+)-limonene and 3% (-)-limonene; the remainder are mostly bicyclic monoterpenes]. This study provides the foundation for a more in-depth understanding of the formation of enantiomeric series of monoterpenes, which can have vastly different olfactory properties.
单萜合酶催化单萜生物合成中的第一个关键步骤，在这一类代谢物的巨大结构多样性中起着一定的作用。在这里，我们研究了结合香叶基二磷酸作为共同底物的柠檬烯合酶中形成柠檬烯对映异构体的结构-功能关系。基于同时考虑晶体结构数据和氨基酸序列差异的分析，我们确定了候选活性位点残基，这些残基可能在催化涉及容纳相反对映体系列反应中间体的反应中发挥作用。我们证明，留兰香（-）-柠檬烯合酶[生成的（-）-柠檬烯多于（+）-柠檬烯]可以通过交换四个残基（C321S、N345I、I453V 和 M458V）转化为突变酶，该突变酶产生具有反转对映选择性的（+）-柠檬烯[80%（+）-柠檬烯和 3%（-）-柠檬烯；其余的主要是双环单萜]。这项研究为更深入地了解单萜对映体系列的形成提供了基础，这些单萜对映体系列可能具有截然不同的嗅觉特性。
