Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production

Philipp Zerbe,Angela Chiang,Macaire Yuen,Björn Hamberger,Britta Hamberger,Jason A Draper,Robert Britton,Jörg Bohlmann

doi:10.1074/jbc.m111.317669

Abstract

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production.

Highlights

Balsam fir produces cis-abienol, a natural product of value to the fragrance industry
We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily
Variations on a common theme of diterpene synthases (diTPSs)-catalyzed cycloisomerization of GGPP 1 contribute substantially to the chemical diversity of diterpene metabolites found in the oleoresin of conifers and in nature in general

Summary

Background

Balsam fir produces cis-abienol, a natural product of value to the fragrance industry. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. A plausible reaction sequence of cis-abienol 4 formation, catalyzed by a bifunctional conifer class I/II diTPS could proceed via water capture of a carbocation intermediate at carbon C-8 and subsequent ionization of the allylic diphosphate group without further cycloisomerization (Fig. 1). A monofunctional angiosperm diTPS catalyzing the formation of a bicyclic oxygen-containing diterpenoid, copal8-ol diphosphate synthase from Cistus creticus (Cistaceae), has been reported [23] This enzyme represents a class II diTPS, which catalyzes the protonation-initiated cyclization of GGPP 1 to form the hydroxylated CPP compound. A novel feature of CAS is the formation of a tertiary diterpenol at the class II active site of a bifunctional class I/II diTPS

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION