Chrysanthemyl diphosphate synthase: Isolation of the gene andcharacterization of the recombinant non-head-to-tail monoterpenesynthase from Chrysanthemum cinerariaefolium

摘要

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium λ 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 plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni²⁺ chromatography. Incubation ofrecombinant CPPase with dimethylallyl diphosphate produced CPP. Thediphosphate ester was hydrolyzed by alkaline phosphatase, and theresulting monoterpene alcohol was analyzed by GC/MS to confirmits structure. The amino acid sequence of CPPase aligns closely withthat of the chain elongation prenyltransferase farnesyl diphosphatesynthase rather than squalene synthase or phytoene synthase, whichcatalyze c1′-2-3 cyclopropanation reactions similar to the CPPasereaction.