The Product Specificities of Terpinolene Synthase, from Cannabis sativa, Reveals the Plasticity of the Terpene Synthase Active Site: supplementary data

Cannabis sativa is a high-value plant renowned for its diverse chemical composition and abundant terpene content, contributing to its unique aroma, flavour, and therapeutic effects. Terpenes significantly influence consumer preference for C. sativa products, driving scientific interest in optimising terpene expression profiles and shaping the selective breeding of terpene profiles in C. sativa cultivars. In particular, the monoterpene, terpinolene, is influential in defining the sensory and therapeutic qualities of many C. sativa strains due to its woody, citrus-like aroma. Here we report the 2.5 Å resolution crystal structure of terpinolene synthase (CsTOS) from C. sativa in its apo form. The structure exhibits the class I monoterpene synthase fold with an open active site conformation. Using site-directed mutagenesis, we identified H618 as a key residues in determining product specificity. Substituting H618 with charged residues resulted in the preferential formation of limonene over terpinolene, highlighting its critical role in stabilising the substrate intermediate. Additionally, novel mutations uncovered an extended epistatic network of residues within 5 Å of the active site, spanning the α-helical bundle of the terpene synthase fold. These interactions contribute to monoterpene formation by modulating substrate positioning and catalytic activity. These insights advance our understanding of monoterpene biosynthesis and enable the targeted engineering of terpene synthases for customised terpene production, offering significant potential for the C. sativa industry.