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 (Ki = 32 ± 9 lM). These results
suggest that crops able to accumulate higher concentration of brassilexin would display higher resistance
levels to the fungus.