MONOTERPENE SYNTHASES FROM GYMNOSPERMS AND ANGIOSPERMS - STEREOSPECIFICITY AND INACTIVATION BY CYSTEINYL-DIRECTED AND ARGINYL-DIRECTED MODIFYING REAGENTS

To further define specific structural and mechanistic differences amon g monoterpene syntheses from divergent plant sources, the stereospecif icity of the enzyme-catalyzed isomerization of geranyl pyrophosphate t o linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which have been well established for monoterpene synthases fr om herbaceous angiosperms) were examined for monoterpene syntheses fro m a conifer, lodgepole pine (Pinus contorta). The chiral monoterpenes isolated from lodgepole pine oleoresin and the major chiral products f rom cell-free assays of each of the four lodgepole pine monoterpene sy nthases belonged to the stereochemical family related by the biosynthe tic intermediacy of 3S-linalyl pyrophosphate. Furthermore, both the pu tative intermediate, 3S-linalyl pyrophosphate, and the natural substra te, geranyl pyrophosphate, were enzymatically converted to the same mo noterpene enantiomers. Thus, like monoterpene synthases from herbaceou s angiosperms, monoterpene syntheses from lodgepole pine appear to cat alyze both the stereospecific isomerization of geranyl pyrophosphate t o linalyl pyrophosphate and the subsequent cyclization of this enzyme- bound intermediate to multiple, stereochemically related monoterpene o lefin isomers. The susceptibility of monoterpene synthases to inactiva tion 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 he rbaceous angiosperms, monoterpene synthases from lodgepole pine were n ot protected against inactivation by coincubation with substrate and m etal ion cofactor. Lodgepole pine monoterpene synthases were also inac tivated by the arginyl-directed reagent phenylglyoxal, and coincubatio n with substrate and cofactor, to effect active-site protection, reduc ed the rate of inactivation 10-fold. (+)-Pinene synthase and (-)-pinen e synthase from sage were also inactivated by phenylglyoxal, but no pr otection was afforded by coincubation with substrate and cofactor. Thu s, monoterpene synthases of conifers appear to have catalytically impo rtant arginyl residues specifically located at or near the active site and have at least some catalytically important thiol residues at a no n-substrate-protectable region of the enzyme, in contrast to monoterpe ne synthases from angiosperms which appear to have catalytically impor tant cysteinyl residues at the active site and have catalytically impo rtant arginyl residues located at a non-substrate-protectable region o f the enzyme. (C) 1995 Academic Press, Inc.