Oxidosqualene cyclases catalyze the biotransformation of acyclic (3S)-2,3-oxidosqualene (OS) to a variety of polycyclic sterols and triterpenoids, generating over 100 distinct triterpenoid skeletons with the formula C 30 H 50 O. [1-3, 4 and references therein] Product specificity is species-dependent and precisely controlled by the prefolded substrate conformation as well as by interactions between the carbocationic intermediate for deprotonation and the functional groups of catalytic amino acid residues of the enzyme. The transformation mechanisms of this single class of enzymes can vary widely. For example, the triterpenes lanosterol, cycloartenol, and parkeol are formed from a preorganized chair-boat-chair substrate conformation of OS, and cationic cyclization to the protosteryl cation is followed by skeletal rearrangements until the final deprotonation step. Formation of the pentacyclic b-amyrin and lupeol proceed similarly except that OS is in the chair-chair-chair conformation (this results in stereochemical differences in the products relative to the chair-boat-chair substrate conformation), and the cationic cyclization to the dammarenyl cation is followed by annulation of a fifth ring.
Various strategies have been used to probe the complex cyclization/rearrangement reaction mechanism, both for the purpose of understanding these complex enzymes and also to engineer cyclases to generate new product profiles. For example, site-directed mutagenesis was used to identify the residues responsible for the product specificity of b-amyrin synthase (PNY) and lupeol synthase (OEW). Two residues of PNY from Panax ginseng, Trp259 and Tyr261, were found to play important roles in the reaction mechanism to direct b-amyrin and/or lupeol formation. [5] We and others independently identified several critical residues from oxidosqualene-lanosterol cyclase (ERG7) from Saccharomyces cerevisiae and oxidosqualenecycloartenol synthase (CAS) from Arabidopsis thaliana, and demonstrated their roles in facilitating tetracyclic formation and/or stabilizing the lanosteryl cation for deprotonation, as