title: Comparative biochemical characterization of nitrile-forming proteins from plants and insects that alter myrosinase-catalysed hydrolysis of glucosinolates.
abstract: The defensive function of the glucosinolate-myrosinase system in plants of the order Capparales results from the formation of isothiocyanates when glucosinolates are hydrolysed by myrosinases upon tissue damage. In some glucosinolate-containing plant species, as well as in the insect herbivore Pieris rapae, protein factors alter the outcome of myrosinase-catalysed glucosinolate hydrolysis, leading to the formation of products other than isothiocyanates. To date, two such proteins have been identified at the molecular level, the epithiospecifier protein (ESP) from Arabidopsis thaliana and the nitrile-specifier protein (NSP) from P. rapae. These proteins share no sequence similarity although they both promote the formation of nitriles. To understand the biochemical bases of nitrile formation, we compared some of the properties of these proteins using purified preparations. We show that both proteins appear to be true enzymes rather than allosteric cofactors of myrosinases, based on their substrate and product specificities and the fact that the proportion of glucosinolates hydrolysed to nitriles does not remain constant when myrosinase activity varies. No stable association between ESP and myrosinase could be demonstrated during affinity chromatography, nevertheless some proximity of ESP to myrosinase is required for epithionitrile formation to occur, as evidenced by the lack of ESP activity when it was spatially separated from myrosinase in a dialysis chamber. The significant difference in substrate- and product specificities between A. thaliana ESP and P. rapae NSP is consonant with their different ecological functions. Furthermore, ESP and NSP differ remarkably in their requirements for metal ion cofactors. We found no indications of the involvement of a free radical mechanism in epithionitrile formation by ESP as suggested in earlier reports.
authors.affiliation: Max Planck Institute for Chemical Ecology, Department of Biochemistry, Jena, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Burow
authors.forename: Jana
authors.initials: J
authors.lastname: Markert
authors.forename: Jonathan
authors.initials: J
authors.lastname: Gershenzon
authors.forename: Ute
authors.initials: U
authors.lastname: Wittstock
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005961:Glucosinolates / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D019476:Insect Proteins / Q000737:chemistry / Q000494:pharmacology*; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009570:Nitriles / Q000494:pharmacology*; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D029681:Arabidopsis Proteins; D005961:Glucosinolates; D019476:Insect Proteins; D009570:Nitriles; D010940:Plant Proteins; D007501:Iron; D006026:Glycoside Hydrolases; C022739:thioglucosidase