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

Abstract

The magnificent chemodiversity of terpenoid natural products is largely rooted in two types of terpene synthases: prenyltransferases and cyclases. The unusual diterpene (C20) 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 C20 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.1 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 C5n 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 C15 prenyltransferase chimera.2 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.