Assembly-line catalysis in bifunctional terpene synthases: (+)-Copalyl diphosphate synthase from fungal Penicillium species.

The magnificent chemodiversity of terpenoid natural products is largely rooted in two types of terpene synthases: prenyltransferases and cyclases. The unusual diterpene (C ) synthase, (+)-copalyl diphosphate synthase from fungal Penicilliumspecies (PvCPS, PfCPS),is the first bifunctional terpene synthase identified with both prenyltransferase and class II cyclase activities in a single polypeptide chain. The C-terminal prenyltransferase α-domain generates the C linear isoprenoid, geranylgeranyl diphosphate, which is then cyclized to (+)-copalyl diphosphate at the interface of the N-terminal βγ-domains. We, first, establish that PvCPS exists as a hexamer - a unique quaternary structure for known αβγ terpene synthases. Hexamer assembly is corroborated by 1) crystal structures of the prenyltransferase α-domain obtained from limited proteolysis of full-length PvCPS, 2) the ab initio modeling of full-length PvCPS derived from small-angle X-ray scattering data, and 3) preliminary 3D reconstructions of PfCPS by cryo-EM. Interestingly, biophysical experiments with PvCPS suggest that oligomer formation is dynamic since the hexamer dissociates into lower-order species at lower concentrations. However, enzyme concentration does not affect prenyltransferase activity in vitro. Assembly-line catalysis provides an efficient carbon management system for generating high-value terpenoid natural products. With the goal of generating different C isoprenoid products, we next explored the structure-function relationship of the prenyltransferase domain within the assembly-line platform of PvCPS. Steady-state kinetics, product analysis, and crystal structures of various structure guided site-directed variants afforded a functional C prenyltransferase chimera. This variant will be used in future engineering experiments to create bifunctional sesquiterpene synthases. REFERENCES: 1. Ronnebaum, T.A.; Gupta, K.; Christianson, D.W., J. Struct. Biol.2020, 210 (1), 107463. 2. Ronnebaum, T.A.; Eaton, S.A.; Brackhahn, E.A.E.; Christianson, D.W., Biochem. 2021, 60 (42), 3162-3272.