The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase

Abstract

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

FIGURE 1.

Schematic representation of IPP and DMAPP coupling reactions catalyzed by typical trans- and cis-PDPS family members. The newly identified enzyme and the reaction it catalyzed are circled (adapted from Thulasiram et al. (3)). CPP, chrysanthemyl diphosphate; CPPS, chrysanthemyl diphosphate synthase; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; GPP, geranyl diphosphate; GPPS, geranyl diphosphate synthase; LPP, lavandulyl diphosphate; LiLPPS, L. x intermedia lavandulyl diphosphate synthase; LinS, linalool synthase; NPP, neryl diphosphate; and NPPS, neryl diphosphate synthase.

FIGURE 2.

Multiple alignment of LiLPPS with NPPS and cis-FPPS of tomato. Bars indicate the five conserved regions (I–V), and the aspartate and glutamate residues in Regions IV and V, respectively, are boxed. Identical amino acid residues are represented by asterisks, conserved amino acid substitutions are represented by a semicolon, and semiconserved amino acid substitutions are represented by a period. LiLPPS (JX985358), L. x intermedia lavandulyl diphosphate synthase; zFPS_SOLHA (B8XA40.1), Z,Z-farnesyl diphosphate synthase from (S. habrochaites) and NPPS (NP_001234633.1), neryl diphosphate synthase from (S. lycopersicum).

FIGURE 3.

GC chromatogram and mass spectrum of LiLPPS product from DMAPP after calf intestinal alkaline phosphatase hydrolysis (A) and authentic lavandulol standard (B). Peaks corresponds to: IPP prenyl alcohol (1), DMAPP prenyl alcohol (2), and lavandulol (3).

FIGURE 4.

LPP accumulation versus DMAPP and IPP consumption (R² = 0.99, p < 0.0001, n = 2). Error bars indicate S.D.

FIGURE 5.

Kinetic properties of LiLPPS at increasing DMAPP concentrations. The nonlinear regression equation used to fit LPP accumulation against DMAPP concentrations was V = V_max*[S]^H/(k_mm^H + [S]^H) (n = 2). Error bars indicate S.D.

FIGURE 6.

Phylogenetic relationship and classification of prenyltransferases. Prenyltransferases within the same class share a minimum of 50% amino acid identity. The trans-PDPS family members are boxed with a broken line, cis-PDPS family members are circled with a solid line, and the prenyltransferase described in this article is boxed with a solid line. The scale bar represents amino acid substitutions per site, and numbers represent the branch support values in percentage. Accession numbers of prenyltransferases used to generate the phylogenetic tree are: AsCPPS, AY308478.1; AtdDPPS1, NP_565551.1; AtdDPPS4, NP_568883.1; AtuDPPS, AAM67372.1; cis-SlFPPS, B8XA40.1; cis-SlPDPS6, AFW98430.1; LiLPPS, JX985358; lsu-MpGPPS, AAF08793.1; lsu-SmGPPS, AEZ55681.1; MpFPPS, AAK63847.1; MtdDPPS, XP_003615624.1; PkGPPS, AAW66658.1; SmFPPS, ABV08819.1; SmGPPS, ACR19637.1; SlGPPS, NP_001234302.1; SlNPPS, NP_001234633.1; ssu-AmGPPS, AAS82859.1; ssu-LiGPPS, JX985359; ssu-MpGPPS, AAF08792.1; ssu-SmGPPS, AEZ55678.1; and TcCPPS, HQ235057.1.