Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity

Liping Gao,Geert Stoopen,Ting Yang,Caiyun Wang,Hao Hu,Maarten A Jongsma

doi:10.1074/jbc.m114.623348

Abstract

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.

Highlights

Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid, chrysanthemyl diphosphate (CPP)
Pyrethrins represent the economically most important and widely used natural pesticide, not much is known about their biosynthesis at the genetic or enzymatic levels
Our in vitro and in vivo results demonstrate that CDS catalyzes the subsequent conversion of CPP to chry

Summary

Background

Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid, chrysanthemyl diphosphate (CPP). CDS, the enzyme catalyzing the first pathway-specific step, evolved from farnesyl diphosphate synthase (FDS) [8, 11, 12] It takes two molecules of DMAPP as substrate to produce CPP as major product and lavandulyl diphosphate (LPP) as minor product (Fig. 1). Phytoene synthase catalyzes a similar reaction, taking geranylgeranyl diphosphate as substrate to give prephytoene diphosphate as an intermediate and phytoene as a final product [16] Both these terpenoid synthases are bifunctional enzymes catalyzing consecutive prenylation and dephosphorylation steps [18, 19]. We report the characterization of CDS to synthesize chrysanthemol from CPP and DMAPP in vitro and the confirmation of this claim in planta using transgenic tobacco plants

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION