Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of t wo molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosph ate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linka ge between isoprenoid units. irregular monoterpenes are common in Chrysanth emum cinerariaefolium and related members of the Asteraceae family. In C. c inerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethri n ester insecticides. CPPase was purified from immature chrysanthemum flowe rs, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plasti dial targeting sequence. Recombinant CPPase bearing an N-terminal polyhisti dine affinity tag in place of the targeting sequence was purified to homoge neity;from an overproducing Escherichia coli strain by Ni2+ chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced C PP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the r esulting monoterpene alcohol was analyzed by GC/MS to confirm its structure . The amino acid sequence of CPPase aligns closely with that of the chain e longation prenyltransferase farnesyl diphosphate synthase rather than squal ene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.