Enzymatic functions of wild tomato methylketone synthases 1 and 2

Abstract

The trichomes of the wild tomato species Solanum habrochaites subsp. glabratum synthesize and store high levels of methylketones, primarily 2-tridecanone and 2-undecanone, that protect the plants against various herbivorous insects. Previously, we identified cDNAs encoding two proteins necessary for methylketone biosynthesis, designated methylketone synthase 1 (ShMKS1) and ShMKS2. Here, we report the isolation of genomic sequences encoding ShMKS1 and ShMKS2 as well as the homologous genes from the cultivated tomato, Solanum lycopersicum. We show that a full-length transcript of ShMKS2 encodes a protein that is localized in the plastids. By expressing ShMKS1 and ShMKS2 in Escherichia coli and analyzing the products formed, as well as by performing in vitro assays with both ShMKS1and ShMKS2, we conclude that ShMKS2 acts as a thioesterase hydrolyzing 3-ketoacyl-acyl carrier proteins (plastid-localized intermediates of fatty acid biosynthesis) to release 3-ketoacids and that ShMKS1 subsequently catalyzes the decarboxylation of these liberated 3-ketoacids, forming the methylketone products. Genes encoding proteins with high similarity to ShMKS2, a member of the "hot-dog fold" protein family that is known to include other thioesterases in nonplant organisms, are present in plant species outside the genus Solanum. We show that a related enzyme from Arabidopsis (Arabidopsis thaliana) also produces 3-ketoacids when recombinantly expressed in E. coli. Thus, the thioesterase activity of proteins in this family appears to be ancient. In contrast, the 3-ketoacid decarboxylase activity of ShMKS1, which belongs to the alpha/beta-hydrolase fold superfamily, appears to have emerged more recently, possibly within the genus Solanum.

Figure 1.

A schematic reaction sequence for the synthesis of straight-chain methylketones. 3-Ketoacyl-ACP or 3-ketoacyl-CoA intermediates of fatty acid synthesis and degradation, respectively, are first hydrolyzed, and the resulting 3-ketoacids are then decarboxylated to give the corresponding 2-methylketone.

Figure 2.

Comparison of the protein sequence of S. habrochaites glabratum ShMKS1 with homologous (MKS1-Like, or MKS1L) sequences from S. lycopersicum, grape (Vv), poplar (Pt), and Arabidopsis (At). Accession numbers are as follows: ShMKS1, GU987105; SlMKS1a, GU987107; SlMKS1b, GU987108; SlMKS1d, GU987110; SlMKS1e, GU987111; PtMKS1L, XM_002313048; VvMKS1L, XM_002284871; AtMES3, At2g23610.

Figure 3.

Comparison of the protein sequence of S. habrochaites glabratum ShMKS2 with homologous sequences from S. lycopersicum, Arabidopsis, and Pseudomonas species (Ps). Accession numbers are as follows: ShMKS2, GU987106; SlMKS2a, GU987112; SlMKS2b, GU9877113; SlMKS2c, GU987114; Ps4HB, EF569604. The initiating Met codon used to produce ShMKS2 protein without the transit peptide is underlined.

Figure 4.

Subcellular localization of ShMKS2-eGFP fusion proteins in N. benthamiana leaf cells. The panels shown on the left exhibit green fluorescence from eGFP, the panels in the middle show red fluorescence from plastidic chlorophyll, and each panel in the right column exhibits an overlay of the two panels to its left. A to C, Tobacco cells infiltrated with an empty binary vector. D to F, Tobacco cells infiltrated with a binary vector carrying the complete opening reading frame of ShMKS2 fused to eGFP. G to I, Tobacco cells infiltrated with a binary vector carrying the ShMKS2 gene lacking the putative transit peptide and fused to eGFP. Bars = 10 μm.

Figure 5.

Total amount of methylketones found in spent medium of E. coli cells expressing ShMKS1, ShMKS2, and ShMKS2(D79A) (all missing the transit peptide-coding region) from the pEXP-TOPO-CT bacterial expression vector. Cells were grown and spent medium was collected and treated as described in “Materials and Methods.” Control 1 cells expressed Clarkia breweri Isoeugenol synthase1 on pEXP5-CT/TOPO (Koeduka et al., 2008). Control 2 cells contained a pEXP5-CT/TOPO vector with no insert. Values are averages ± se calculated from three experiments.

Figure 6.

Methylketone production by E. coli cells expressing ShMKS2. Treated and nontreated spent medium of E. coli cells expressing ShMKS2 (without the transit peptide-coding region) was extracted with hexane, and the methylketone content was measured by GC-MS. Treatments included heat, acid and heat, purified ShMKS1 protein in phosphate buffer, and phosphate buffer alone. Values are averages ± se calculated from three experiments. See text for details.

Figure 7.

Decarboxylase activity assays for ShMKS1 and ShMKS2 using 3-ketomyristic acid as the substrate. Purified recombinant proteins were assayed as described in “Materials and Methods,” and the mean and sd values were calculated from three replicates and given as shown.

Figure 8.

Thioesterase activity assays for ShMKS1 and ShMKS2. To a 500-μL solution of enzymatically prepared 3-ketomyristoyl-ACP (see “Materials and Methods”), a 20-μL solution of the following was added: lane 1, enzyme buffer; lane 2, 2.5 μg of ShMKS1 in buffer; lane 3, enzyme buffer; lane 4, 2.5 μg of ShMKS2; lane 5, 2.5 μg of ShMKS1 and 2.5 μg of ShMKS2. Each reaction was incubated for 30 min at 23°C, after which the reaction solution was either extracted directly with hexane (lanes 1, 2, and 5) or first treated with acid and heated at 75°C for 30 min (lanes 3 and 4), then cooled down to room temperature and extracted with hexane. Hexane extracts were analyzed by GC-MS. Mean and sd values were calculated from three replicates.