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Blackleg (Leptosphaeria maculans and Leptosphaeria biglobosa) and black spot (Alternaria brassicicola) fungi are devastating plant pathogens known to detoxify the plant defence metabolite, brassinin. The significant roles of brassinin as a crucifer phytoalexin and as a biosynthetic precursor of several other plant defences make it important in plant fitness. Brassinin detoxifying enzymes produced by L. maculans and A. brassicicola catalyse the detoxification of brassinin by hydrolysis of its dithiocarbamate group to indolyl-3-methanamine. The purification and characterization of brassinin hydrolases produced by L. maculans (BHLmL2) and A. brassicicola (BHAb) were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa. Protein characterization using LC-MS/MS and sequence alignment analyses suggested that both enzymes belong to the family of amidases with the catalytic Ser/Ser/Lys triad. Furthermore, chemical modification of BHLmL2 and BHAb with selective reagents suggested that the amino acid serine was involved in the catalytic activity of both enzymes. The overall results indicated that BHs have new substrate specificities with a new catalytic activity that can be designated as dithiocarbamate hydrolase. Investigation of the effect of various phytoalexins on the activities of BHLmL2 and BHAb indicated that cyclobrassinin was a competitive inhibitor of both enzymes. On the basis of pH dependence, sequence analyses, chemical modifications of amino acid residues and identification of headspace volatiles, a chemical mechanism for hydrolysis of the dithiocarbamate group of brassinin catalysed by BHLmL2 and BHAb is proposed. The current information should facilitate the design of specific synthetic inhibitors of these enzymes for plant treatments against blackleg and black spot fungal infections.