Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases

Abstract

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αβγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [Chen, M., Chou, W. K. W., Toyomasu, T., Cane, D. E., Christianson, D. W. (2016) ACS Chem. Biol. 11, 889–899]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20 diterpene products and three C25 sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.

Chimeric Terpene Synthases Possessing both Terpene Cyclization and Prenyltransfer Activities

“…For example, am utated FgMS (I129F or N311F) becomes aC 15 -specific enzyme, although the wild-type FgMS can utilize FPP,G GPP,a nd GFPP. [40] These experiments suggested that only af ew amino acid resi-dues regulate the substrate specificityo ft he TPS domain. [39,40] However,t he exchange of only two amino acid residues (W225L/V228A) in its typeI TPS active site pocket enables its TPS domain to utilize (C 25 )G FPP and produce sesterterpenes.…”

“…[29] The last difference is the degree of interdependence between the a and bg domains. [40] TbTS requires both the a and b domains for its activity,a lthough its actives ite exists solely in the a domain. [30] This feature is dissimilar from the other abg TPSs.…”

“…[29j] Another example is PaPS, which can only accept up to C 20 linear substrates. [39,40] However,t he exchange of only two amino acid residues (W225L/V228A) in its typeI TPS active site pocket enables its TPS domain to utilize (C 25 )G FPP and produce sesterterpenes. [40] These experiments suggested that only af ew amino acid resi-dues regulate the substrate specificityo ft he TPS domain.…”

Engineering isoprene synthesis in cyanobacteria

“…There are over 80 000 different known terpenoids, with the number of defined structures doubling every decade since the 1970s [20,21]. There are over 80 000 different known terpenoids, with the number of defined structures doubling every decade since the 1970s [20,21].…”

Identification of Chimeric αβγ Diterpene Synthases Possessing both Type II Terpene Cyclase and Prenyltransferase Activities

“…For example, AgAS, the abietadiene synthase from Abies grandis (αβγ bifunctional type I+type II TC), shows high interdependence between the two catalytic domains because the separate α and βγ domain constructs are both catalytically inactive . Additionally, in the case of TbTS, the taxadiene synthase from Taxus brevifolia (αβγ monofunctional type I TC), both the α and β domains are necessary for its activity, despite the fact that its active site exists solely in the α domain . In the case of FCPS/KS, the ent‐kaurene synthase from Phaeosphaeria sp.…”