title: 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.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
authors.forename: Geng
authors.initials: G
authors.lastname: Yu
authors.forename: Thuong T H
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Yongxia
authors.initials: Y
authors.lastname: Guo
authors.forename: Ines
authors.initials: I
authors.lastname: Schauvinhold
authors.forename: Michele E
authors.initials: ME
authors.lastname: Auldridge
authors.forename: Nazmul
authors.initials: N
authors.lastname: Bhuiyan
authors.forename: Imri
authors.initials: I
authors.lastname: Ben-Israel
authors.forename: Yoko
authors.initials: Y
authors.lastname: Iijima
authors.forename: Eyal
authors.initials: E
authors.lastname: Fridman
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: Eran
authors.initials: E
authors.lastname: Pichersky
mesh_terms: D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D007659:Ketones; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C009541:2-tridecanone; D004950:Esterases; D002262:Carboxy-Lyases; C526928:undecan-2-one