Migrated Hopene Synthases from Colysis pothifolia and Identification of a Migration Switch Controlling the Number of 1,2-Hydride and Methyl Shifts.

Abstract

Ferns are known to produce triterpenes, derived from squalene, that are synthesized by squalene cyclases (SCs). Among these, Colysis species produce onoceroids, the bis-cyclic skeleton of which can be cyclized from both termini of squalene. To gain insight into the molecular basis of triterpene structural diversity, cDNA cloning of SCs from C. elliptica and C. pothifolia was performed; this leads to the isolation of five SC cDNAs. Functional analysis of these clones revealed their enzymatic products to be hop-22(29)-ene, α-polypodatetraene, and hop-17(21)-ene. One of these clones (CPF) is a transcribed pseudogene with a 22-nucleotide deletion causing a nonsense mutation. To predict the inherent function of CPF, we constructed an insertion mutant of CPF that successfully converted inert CPF to the active SC, the product of which is fern-9(11)-ene. Subsequent mutations identified active-site residues that control the number of 1,2-hydride and methyl shifts.