Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site. (May 2017)

• Record Type: Journal Article
• Authors: Xu, Jinkun; Ai, Ying; Wang, Jianhui; Xu, Jingwei; Zhang, Yongkang; Yang, Dong
• Is Part Of: Phytochemistry. Volume 137(2017)
• Page Start: 34
• Page End: 41
• Publication Date: 2017-05
• Subjects: Limonene -- Pinene -- Phellandrene -- Terpene synthase -- Evolution -- Site-directed mutagenesis -- Terpene diversity; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2017.02.017
• Languages: English
• ISSNs: 0031-9422

Abstract

Abstract: S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases. Graphical abstract: Mutation N345A/L423A/S454A converts M. spicata limonene synthase to a pinene synthase, while a single mutation N345I converts it to a phellandrene synthase. Polar residues in the active pocket may be essential for limonene production by stabilizing the terpinyl cation. Highlights: Limonene synthase is converted to pinene synthase by three mutations. Limonene synthase is converted to phellandrene synthase by a single mutation. The polarity of the residue 345 is critical for limonene synthesis. Polar residues may form a "polar pocket" to stabilize the terpinyl cation.