Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium
Sb. Rivera et al., Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium, P NAS US, 98(8), 2001, pp. 4373-4378
Citations number
48
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.