title: The phytoalexins brassilexin and camalexin inhibit cyclobrassinin hydrolase, a unique enzyme from the fungal pathogen Alternaria brassicicola.
abstract: Alternaria brassicicola is a fungal pathogen of many agriculturally important cruciferous crops. Cyclobrassinin hydrolase (CH) is an enzyme produced by A. brassicicola that catalyzes the transformation of the cruciferous phytoalexin cyclobrassinin into S-methyl[(2-sulfanyl-1H-indolyl-3)methyl]carbamothioate. The purification and characterization of CH was performed using a four-step chromatography method. SDS-PAGE and gel exclusion chromatography indicated that CH is a tetrameric protein with molecular mass of 330 kDa. Sequence analysis and chemical modification of CH with selective reagents suggested that the enzyme mediates hydrolysis of cyclobrassinin using a catalytic amino acid triad. Enzyme kinetic studies using cyclobrassinin and 1-methylcyclobrassinin as substrates revealed that CH displayed positive substrate cooperativity. Investigation of the effect of nine phytoalexins and two derivatives on the activity of CH indicated that six compounds displayed inhibitory activity: brassilexin, 1-methylbrassilexin, dioxibrassinin, camalexin, brassicanal A and sinalexin. The enzyme kinetics of CH strongly suggested that brassilexin and 1-methylbrassilexin are noncompetitive inhibitors of CH activity, and that camalexin is a competitive inhibitor while dioxibrassinin inhibits CH through a mixed mechanism. The phytoalexin brassilexin is the most effective inhibitor of CH (K(i)=32 ± 9 μM). These results suggest that crops able to accumulate higher concentration of brassilexin would display higher resistance levels to the fungus.
authors.affiliation: Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada. Electronic address: s.pedras@usask.ca.
authors.forename: M Soledade C
authors.initials: MS
authors.lastname: Pedras
authors.affiliation: Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Minic
mesh_terms: D000528:Alternaria / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism; D005656:Fungal Proteins / Q000032:analysis; D007211:Indoles / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000494:pharmacology; D012717:Sesquiterpenes / Q000737:chemistry*; D013172:Spores, Fungal / Q000254:growth & development; D013844:Thiazoles / Q000737:chemistry* / Q000494:pharmacology; D013859:Thiocarbamates / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D000094162:Phytoalexins
chemical_list: D005656:Fungal Proteins; D007211:Indoles; D012717:Sesquiterpenes; D013844:Thiazoles; D013859:Thiocarbamates; C102405:camalexin; C581768:cyclobrassinin; C430121:brassilexin; D000094162:Phytoalexins
keywords: Brassicanal A; Brassilexin; Brassinin; Camalexin; Crucifer; Cyclobrassinin; Dioxibrassinin; Hydrolase; Paldoxin; Phytoalexin; Sinalexin