Nitrilase protein families in glucosinolate forming plants

MARKUS PIOTROWSKI
Ruhr-Universität Bochum
Lehrstuhl für Pflanzenphysiologie
ND3
Universitätsstraße 150
D-44801 Bochum
Markus.Piotrowski@ruhr-uni-bochum.de
http://www.ruhr-uni-bochum.de/pflaphy/Seiten/PG_Piotrowski_engl.html

References
Piotrowski, M., Schemenewitz, A., Lopukhina, A., Müller, A., Janowitz, T., Weiler, E.W., and Oecking, C. (2004) Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyzing the final step in biosynthesis of the glucosinolate core structure. J. Biol. Chem., 279:50717-50725.
The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.

Piotrowski, M. and Volmer, J.J. (2006) Cyanide metabolism in higher plants: Cyanoalanine hydratase is a NIT4 homolog. Plant Mol. Biol., 61:111-122.
Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of b-cyano-L-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called 'cyanoalanine hydratase' of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.

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