title: A thiocyanate-forming protein generates multiple products upon allylglucosinolate breakdown in Thlaspi arvense.
abstract: Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate-myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe²⁺ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution.
authors.affiliation: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig, Germany.
authors.forename: Jennifer-C
authors.initials: JC
authors.lastname: Kuchernig
authors.forename: Anita
authors.initials: A
authors.lastname: Backenköhler
authors.forename: Maike
authors.initials: M
authors.lastname: Lübbecke
authors.forename: Meike
authors.initials: M
authors.lastname: Burow
authors.forename: Ute
authors.initials: U
authors.lastname: Wittstock
mesh_terms: D000498:Allyl Compounds / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005296:Ferrous Compounds; D015870:Gene Expression; D005961:Glucosinolates / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D035261:Plant Components, Aerial / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018517:Plant Roots / Q000737:chemistry; D000644:Quaternary Ammonium Compounds; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013861:Thiocyanates / Q000737:chemistry / Q000378:metabolism*; D031227:Thlaspi / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D000498:Allyl Compounds; D018076:DNA, Complementary; D005296:Ferrous Compounds; D005961:Glucosinolates; D009570:Nitriles; D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; D018749:RNA, Plant; D011994:Recombinant Proteins; D013861:Thiocyanates; C491148:allyl thiocyanate; C038178:ammonium ferrous sulfate; D006026:Glycoside Hydrolases; C022739:thioglucosidase