The phytoalexins brassilexin and camalexin inhibit cyclobrassinin hydrolase,a unique enzyme from the fungal pathogen Alternaria brassicicola |
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| Affiliation: | 1. INSERM U914 ‘Emerging Resistance to Antibiotics’, K.-Bicêtre, France;2. Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland;3. International Center for Medical Research and Training, CIDEIM, Cali, Colombia;1. Chemistry Department, University of Milan, via Golgi 19, 20133 Milano, Italy;2. I-LAMP and Mathematics and Physics Department, Catholic University, via dei Musei 41, 25121 Brescia, Italy;3. CNR – National Research Council of Italy, ISTM, via Golgi 19, 20133 Milan, Italy;1. Minase Research Institute, Ono Pharmaceutical Co., Ltd, Osaka, Japan;2. Department of Immunopharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan;1. Department of Anesthesiology, New York University School of Medicine, New York, NY 10016, USA;2. Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA;1. Radiation Safety Directorate, Partizanski odredi 143, P.O. Box 22, 1020 Skopje, Macedonia;2. Faculty of Natural Sciences and Mathematics, Institute of Mathematics, Saints Cyril and Methodius University, 1000 Skopje, Macedonia |
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| 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 (Ki = 32 ± 9 μM). These results suggest that crops able to accumulate higher concentration of brassilexin would display higher resistance levels to the fungus. |
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| Keywords: | Brassicanal A Brassilexin Brassinin Camalexin Crucifer Cyclobrassinin Dioxibrassinin Hydrolase Paldoxin Phytoalexin Sinalexin |
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