Monoterpene synthases of Pinus contorta and related conifers. A new class of terpenoid cyclase

Abstract

A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand fir (Abies grandis) and other higher plants in molecular mass (67 +/- 2 kDa as estimated by size-exclusion chromatography), Km for geranyl pyrophosphate (7.8 +/- 1.9 microM), and isoelectric point (4.75 +/- 0.2 as determined by isoelectric focusing), but the cyclases from both lodgepole pine and grand fir are unlike previously characterized terpenoid cyclases from angiosperms and fungi, in that they have an alkaline pH optimum (pH 7.8), are activated by K+, Rb+, Cs+, or NH+4 (Li+ and Na+ are not effective), require either Mn2+ or Fe2+ as divalent metal ion cofactors (Mg2+ is not effective), and are not protected by the substrate-metal ion complex against inhibition by the histidine-directed reagent diethyl pyrocarbonate. Chromatography of the pine xylem extracts on a quaternary amino anion-exchange resin results in the separation of four similar, but distinct, multiple product monoterpene synthases that produce sabinene, beta-phellandrene, 3-carene, and beta-pinene as the principal components, respectively. The major cyclase (phellandrene synthase) was subsequently purified by hydroxyapatite chromatography and electrophoresis. V8 proteolysis provided a peptide map significantly different from that obtained with limonene synthase from spearmint (Mentha spicata), and limited NH2-terminal sequencing of the phellandrene synthase fragments revealed no significant similarity to the deduced amino acid sequence of the angiosperm limonene synthase, the only monoterpene cyclase to be cloned and sequenced thus far. Furthermore, polyclonal antibodies raised against the angiosperm limonene synthase did not detectably cross-react with any proteins in extracts from either lodgepole pine or grand fir by immunoblotting analysis. In addition to these structural differences between cyclases from conifers and herbaceous angiosperms, the unusual pH optimum, mono- and divalent metal ion requirement, and reactivity toward histidine carbethoxylation indicate that monoterpene cyclases isolated from conifers may also have a different complement of active-site amino acid residues involved in substrate binding and catalysis than those of terpenoid cyclases previously isolated from angiosperms.