The unusual diterpene (C20) synthase copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is the first bifunctional terpene synthase identified with both prenyltransferase and class II cyclase activities in a single polypeptide chain with domain architecture. The C-terminal prenyltransferase -domain generates geranylgeranyl diphosphate which is then cyclized to form copalyl diphosphate at the interface of the N-terminal -domains. Upon recombinant expression and purification, we now demonstrate that PvCPS exists as a hexamer at high concentrations – a unique quaternary structure for known terpene synthases. Hexamer assembly is corroborated by a 2.41 Å-resolution crystal structure of the -domain prenyltransferase obtained from limited proteolysis of full-length PvCPS, as well as the ab initio molecular envelope of full-length PvCPS derived from small-angle X-ray scattering data. However, the results of size-exclusion chromatography in-line with multiangle light scattering, sedimentation velocity analytical ultracentrifugation, and native polyacrylamide gel electrophoresis experiments suggest that oligomerization is concentration-dependent, such that the hexamer dissociates into individual monomers at lower concentrations. Further investigation indicates PvCPS oligomer assembly does not affect prenyltransferase activity in vitro.

Concentration-dependent oligomer assembly of a chimeric  bifunctional diterpene synthase with prenyltransferase and class II cyclase activities

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.