Mutational Studies on Triterpene Synthases:  Engineering Lupeol Synthase into β-Amyrin Synthase

Abstract

Site-directed mutagenesis was carried out on two triterpene synthases, β-amyrin (PNY) and lupeol (OEW) synthases, to identify the amino acid residues responsible for their product specificity. In addition to sequence comparison among known oxidosqualene cyclases, our previous chimeric studies suggested that 258 MWCYCR 263 sequence of β-amyrin synthase PNY ( 255 MLCYCR 260 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 ∆ 18 ) 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.

Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases from Withania somnifera (L.) Dunal

“…Whereas one of the conserved motif (Motif B) was detected exclusively in WsOSC/BS and WsOSC/LS. Interestingly, on the basis of site-directed mutagenesis, tryptophan and leucine positioned at the second place in these motifs have been proved to be characteristic for functional ␤-amyrin and lupeol synthases (44). However, a point mutation can radically mutate such specificities, but in many situations numerous other sequence alterations may counterbalance each other without modifying the enzyme specificity (45).…”

Molecular Cloning, Functional Expression and Characterization of (E)-.BETA.-Farnesene Synthase from Citrus junos.

“…In fact, substitution of the Tyr261 of b-amyrin synthase, which produces the pentacyclic oleanane skeleton, to a histidine residue by site-directed mutagenesis resulted in the formation of compounds with a tetracyclic dammarane skeleton. 22) To clarify the molecular mechanism of terpene cyclization, site-directed mutagenesis of CJFS and further cloning of terpene synthase genes from a variety of plant species are now underway in our laboratory.…”

Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding β-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase

“…SvBS possesses the amino acid motif DCTAE, thought to form part of the active site of OSC (Abe et al, 1993;Abe and Prestwich, 1995) and the four QW motifs characteristic of the OSC superfamily (Poralla et al, 1994). In addition, SvBS amino acid sequence contains the Trp residue in the MWCYCR motif that plays an important role in the formation of b-amyrin in Panax ginseng (Kushiro et al, 2000).…”