Project Summary/Abstract Oligomeric assembly of terpene synthases improves biosynthetic product flux by proximity or cluster channeling. The unusual diterpene (C20) synthase, PvCPS, from Penicillium verruculosum was identified and characterized as the first bifunctional terpene synthase with abg domain architecture containing both prenyltransferase and class II cyclase activities. The C-terminal a domain generates geranylgeranyl diphosphate which is then cyclized to copalyl diphosphate at the interface of the N-terminal bg domains. Upon recombinant expression and purification, I determined that PvCPS exists as a hexamer at high concentrations – a unique quaternary structure for known abg terpene synthases – and dissociates to monomers at low concentrations. Interestingly, oligomerization is common among prenyltransferases and terpene synthases, which have been observed as monomers, dimers, trimers, tetramers, and hexamers. The first part of this proposal aims to determine the structural aspects of oligomer formation using the prenyltransferase-cyclase chimera of PvCPS by determining the high-resolution structure using Cryo-Electron Microscopy (Cryo-EM). The architecture of PvCPS presents an ideal engineering opportunity for multifunctional assembly-line terpene biosynthesis. Thus, the second part of this proposal aims to utilize the oligomer assembly of PvCPS to engineer novel multifunctional enzymes, since many terpene products exhibit useful pharmaceutical properties. Successful completion of these aims will lead to the development of multifunctional synthases for in vitro biosynthesis of complex terpenes. Lastly, human prenyltransferases, such as farnesyl diphosphate synthase (hFPPS), geranylgeranyl diphosphate synthase (hGGPPS), and squalene synthase (hSQS), have been implicated in a variety of diseases and cancers. Each of these prenyltransferases has been characterized as an oligomer in vitro. However, if oligomer assembly is concentration-dependent, does oligomer assembly occur under cellular conditions? And if so, what is its role? The last aim will determine if oligomer assembly occurs in vivo, and its associated function.