Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Biochemistry. 2023 Aug 1;62(15):2301-2313. doi: 10.1021/acs.biochem.3c00247. Epub 2023 Jul 14.
The class I sesquiterpene cyclase -isozizaene synthase from (EIZS) catalyzes the transformation of linear farnesyl diphosphate (FPP) into the tricyclic hydrocarbon -isozizaene in the biosynthesis of albaflavenone antibiotics. The active site cavity of EIZS is largely framed by four aromatic residues - F95, F96, F198, and W203 - that form a product-shaped contour, serving as a template to chaperone conformations of the flexible substrate and multiple carbocation intermediates leading to -isozizaene. Remolding the active site contour by mutagenesis can redirect the cyclization cascade away from -isozizaene biosynthesis to generate alternative sesquiterpene products. Here, we present the biochemical and structural characterization of four EIZS mutants in which aromatic residues have been substituted with polar residues (F95S, F96H, F198S, and F198T) to generate alternative cyclization products. Most notably, F95S EIZS generates a mixture of monocyclic sesquiterpene precursors of bisabolane, a D2 diesel fuel substitute. X-ray crystal structures of the characterized mutants reveal subtle changes in the active site contour showing how each aromatic residue influences the chemistry of a different carbocation intermediate in the cyclization cascade. We advance that EIZS may serve as a robust platform for the development of designer cyclases for the generation of high-value sesquiterpene products ranging from pharmaceuticals to biofuels in synthetic biology approaches.
（EIZS）类倍半萜环化酶 -异泽兰烯合酶催化线性法呢基二磷酸（FPP）转化为在阿尔巴黄酮抗生素生物合成中的三环烃 -异泽兰烯。EIZS 的活性位点腔主要由四个芳香族残基 - F95、F96、F198 和 W203 - 构成，这些残基形成产物形状的轮廓，作为模板引导柔性底物和导致 -异泽兰烯的多个碳正离子中间物的构象。通过突变重塑活性位点轮廓可以使环化级联偏离 -异泽兰烯生物合成，从而产生替代的倍半萜产物。在这里，我们介绍了四个 EIZS 突变体的生化和结构特征，其中芳香族残基已被极性残基（F95S、F96H、F198S 和 F198T）取代，以产生替代的环化产物。值得注意的是，F95S EIZS 生成了双环倍半萜前体的混合物，双环倍半萜是 D2 柴油燃料的替代品。表征突变体的 X 射线晶体结构显示活性位点轮廓的细微变化，表明每个芳香族残基如何影响环化级联中不同碳正离子中间体的化学性质。我们认为，EIZS 可以作为一种强大的平台，用于开发用于生成高价值倍半萜产物的设计环化酶，这些产物从药物到生物燃料，涵盖了合成生物学方法。
