MONOTERPENE SYNTHASES OF PINUS-CONTORTA AND RELATED CONIFERS - A NEW CLASS OF TERPENOID CYCLASE

A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-H-3(1)]geranyl pyrophosphate to a varie ty of moneterpene olefins found in lodgepole pine oleoresin. This mono terpene synthase activity is similar to previously described terpenoid cyclases from grand fir (Abies grandis) and other higher plants in mo lecular mass (67 +/- 2 kDa as estimated by size exclusion chromatograp hy), K-m for geranyl pyrophosphate (7.8 +/- 1.9 mu M), and isoelectric point (4.75 +/- 0.2 as determined by isoelectric focusing), but the c yclases from both lodgepole pine and grand fir are unlike previously c haracterized terpenoid cyclases from angiosperms and fungi, in that th ey have an alkaline pH optimum (pH 7.8), are activated by K+, Rb+, Cs, or NH4+ (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 substratemetal ion complex against inhibition by the histidine-directed reagent diethyl pyrocarbonate. Chromatography of th e pine xylem extracts on a quaternary amino anion-exchange resin resul ts in the separation of four similar, but distinct, multiple product m onoterpene synthases that produce sabinene, beta-phellandrene, 3-caren e, and beta-pinene as the principal components, respectively. The majo r cyclase (phellandrene synthase) was subsequently purified by hydroxy apatite chromatography and electrophoresis. V8 proteolysis provided a peptide map significantly different from that obtained with limonene s ynthase from spearmint (Mentha spicata), and limited NH2-terminal sequ encing of the phellandrene synthase fragments revealed no significant similarity to the deduced amino acid sequence of the angiosperm limone ne synthase, the only monoterpene cyclase to be cloned and sequenced t hus far. Furthermore, polyclonal antibodies raised against the angiosp erm 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 cyclase s from conifers and herbaceous angiosperms, the unusual pH optimum, mo no- and divalent metal ion requirement, and reactivity toward histidin e carbethoxylation indicate that monoterpene cyclases isolated from co nifers may also have a different complement of active-site amino acid residues involved in substrate binding and catalysis than those of ter penoid cyclases previously isolated from angiosperms.