| Chrysanthemum cinerariaefolium chrysanthemyl diphosphate synthase (CPP synthase) is an irregular, or non-head-to-tail, monoterpene synthase important in the biosynthesis of the industrially useful pyrethrin ester insecticides. The gene for CPP synthase was isolated from a C. cinerariaefolium cDNA library and subcloned into an E. coli overexpression system. Recombinant CPP synthase bearing an N-terminal polyhistidine affinity tag was purified to homogeneity by Ni2+ chromatography and used to determine the products and their structures. When CPP synthase was incubated with dimethylallyl diphosphate (DMAPP), two non-head-to-tail or irregular monoterpenes were produced. The first, chrysanthemyl diphosphate (CPP), contained a c1'-2-3 linkage between isoprenoid units whereas the second product, lavandulyl diphosphate (LPP), displayed a 1'-2 fusion between the DMAPP molecules. The diphosphate esters were hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohols were analyzed by GC/MS to confirm their structures. The stereochemical course of the reaction that yields CPP was investigated with (R) and (S)-[1-2H]-DMAPP. This elimination reaction proceeded with the same stereo selectivity as the biosynthesis of the cyclopropylcarbinyl intermediates produced by squalene synthase and phytoene synthase. These chemically related reactions are important in sterol and carotenoid biosynthesis, respectively. Surprisingly, the CPP synthase sequence shares striking similarity with that of the head-to-tail chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase. These observations indicate that irregular terpenoid synthases have evolved convergently. |
