Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.
From Business Unit PRI-Bioscience, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands, the Laboratory of Entomology, Wageningen UR, P.O. Box 8031, 6700 EH Wageningen, The Netherlands, the Laboratory of Plant Physiology, Wageningen UR, P.O. Box 658, 6700 AR Wageningen, The Netherlands, and.
Ting
T
Yang
From Business Unit PRI-Bioscience, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Liping
L
Gao
the Key Laboratory for Biology of Horticultural Plants, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China.
Hao
H
Hu
From Business Unit PRI-Bioscience, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Geert
G
Stoopen
the Key Laboratory for Biology of Horticultural Plants, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China.
Caiyun
C
Wang
From Business Unit PRI-Bioscience, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands, maarten.jongsma@wur.nl.
Maarten A
MA
Jongsma
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways; D011723:Chrysanthemum cinerariifolium / Q000201:enzymology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000502:physiology*; D030821:Plants, Genetically Modified / Q000235:genetics; D013729:Terpenes / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics
D010940:Plant Proteins; D013729:Terpenes; C056121:chrysanthemol; D019883:Alkyl and Aryl Transferases
Biosynthesis; Enzyme Kinetics; Mutagenesis; Plant Biochemistry; Terpenoid