R. Croteau

Monoterpene synthases from gymnosperms and angiosperms: stereospecificity and inactivation by cysteinyl- and arginyl-directed modifying reagents.

To further define specific structural and mechanistic differences among monoterpene synthases from divergent plant sources, the stereospecificity of the enzyme-catalyzed isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which have been well established for monoterpene synthases from herbaceous angiosperms) were examined for monoterpene synthases from a conifer, lodgepole pine (Pinus contorta). The chiral monoterpenes isolated from lodgepole pine oleoresin and the major chiral products from cell-free assays of each of the four lodgepole pine monoterpene synthases belonged to the stereochemical family related by the biosynthetic intermediacy of 3S-linalyl pyrophosphate. Furthermore, both the putative intermediate, 3S-linalyl pyrophosphate, and the natural substrate, geranyl pyrophosphate, were enzymatically converted to the same monoterpene enantiomers. Thus, like monoterpene synthases from herbaceous angiosperms, monoterpene synthases from lodgepole pine appear to catalyze both the stereospecific isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound intermediate to multiple, stereochemically related monoterpene olefin isomers. The susceptibility of monoterpene synthases to inactivation by cysteinyl- and arginyl-directed chemical modification reagents was also examined to identify specific structural differences between enzymes from conifers and angiosperms. Like monoterpene synthases from peppermint (Mentha x piperita) and culinary sage (Salvia officinalis), monoterpene synthases from lodgepole pine were inactivated by thiol-directed reagents; however, unlike monoterpene synthases from these herbaceous angiosperms, monoterpene synthases from lodgepole pine were not protected against inactivation by coincubation with substrate and metal ion cofactor. Lodgepole pine monoterpene synthases were also inactivated by the arginyl-directed reagent phenylglyoxal, and coincubation with substrate and cofactor, to effect active-site protection, reduced the rate of inactivation 10-fold. (+)-Pinene synthase and (-)-pinene synthase from sage were also inactivated by phenylglyoxal, but no protection was afforded by coincubation with substrate and cofactor. Thus, monoterpene synthases of conifers appear to have catalytically important arginyl residues specifically located at or near the active site and have at least some catalytically important thiol residues at a non-substrate-protectable region of the enzyme, in contrast to monoterpene synthases from angiosperms which appear to have catalytically important cysteinyl residues at the active site and have catalytically important arginyl residues located at a non-substrate-protectable region of the enzyme.

Biosynthesis of the sesquiterpene patchoulol from farnesyl pyrophosphate in leaf extracts of Pogostemon cablin (patchouli): mechanistic considerations.

Several mechanistic alternatives have been proposed for the enzyme-catalyzed, electrophilic cyclization of farnesyl pyrophosphate to the tricyclic sesquiterpene alcohol patchoulol, which is the characteristic component of the essential oil of Pogostemon cablin (patchouli). These alternatives include schemes involving deprotonation-reprotonation steps and the intermediacy of the monocyclic and bicyclic olefins germacrene and bulnesene, respectively, and involving a 1,3-hydride shift with only tertiary cationic intermediates and without any deprotonation-reprotonation steps. Analytical studies, based on analyses of P. cablin leaf oil at different stages of plant development, and in vivo time-course investigations, using 14CO2 and [14C]sucrose, gave no indication that germacrene and bulnesene were intermediates in patchoulol biosynthesis. A soluble enzyme system from P. cablin leaves was prepared, which was capable of converting farnesyl pyrophosphate to patchoulol, and isotopic dilution experiments with both labeled and unlabeled olefins were carried out with this system to confirm that sesquiterpene olefins did not participate as fre intermediates in the transformation of the acyclic precursor to patchoulol. Patchoulol derived biosynthetically from [12,13-14C;1-3H]farnesyl pyrophosphate was chemically degraded to establish the overall construction pattern of the product. Similar studies with [12,13-14C;6-3H]farnesyl pyrophosphate as a precursor eliminated deprotonation steps to form bound olefinic intermediates in the biosynthesis of patchoulol, while providing supporting evidence for the hydride shift mechanism.