Mutational studies on triterpene synthases: Engineering lupeol synthase into β-amyrin synthase

Site-directed mutagenesis was carried out on two triterpene synthases, α-amyrin (PNY) and lupeol (OEW) synthases, to identify the amino acid residues responsible for their product specificity. In addition to sequence comparison among known oxidosqualen cyclases, our previous chimeric studies suggested that ²⁵⁸MWCYCR²⁶³ sequence of β-amyrin synthase PNY (²⁵⁵MLCYCR²⁶⁰ sequence of lupeol synthase OEW) would participate in product differentiation. To test this hypothesis, Trp259 (MWCYCR of PNY) was mutated to Leu (PNY W259L mutant). Functional expression in yeast and product analysis revealed that this mutant produced lupeol as a major product together with β-amyrin in 2:1 ratio. Some other minor products including butyrospermol were also produced. On the other hand, Leu256 (MLCYCR of OEW) was mutated to Trp (OEW L256W mutant). This mutant produced exclusively β-amyrin with only minor amount of lupeol, demonstrating that a single mutation had engineered lupeol synthase into β-amyrin synthase. Therefore, Trp259 of β-amyrin synthase was identified to be the residue controlling β-amyrin formation presumably through stabilization of oleanyl cation, while lack of this effect by Leu residue may terminate the reaction at lupenyl cation stage. In further mutation studies, Tyr residue (MWCYCR in PNY and MLCYCR in OEW) conserved in all of the OSCs producing pentacyclic triterpenes was mutated into His which is found in all of those producing tetracyclic carbon skeletons to investigate the role of this Tyr261 of PNY. PNY Y261H mutant produced dammara-18,21-dien-3β-ol (as a 3:5 mixture of E/Z isomer at Δ¹⁸) together with a minor amount of dammara-18(28),21-dien-3β-ol, demonstrating that Tyr261 of β-amyrin synthase plays an important role in producing pentacyclic triterpenes presumably by stabilizing one of the cation intermediates generated after dammarenyl cation.