Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study
2010
Kopecny, David | Briozzo, Pierre | Popelkova, Hana | Sebela, Marek | Koncitikova, Radka | Spichal, Lukas | Nisler, Jaroslav | Madzak, Catherine | Frebort, Ivo | Laloue, Michel | Houba-Hérin, Nicole | Unité de biologie cellulaire ; Institut National de la Recherche Agronomique (INRA) | Faculty of Science, Department of Biochemistry ; Palacky University Olomouc | Institut Jean-Pierre Bourgin (IJPB) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech | Department of Molecular, Cellular and Developmental Biology ; University of Michigan [Ann Arbor] ; University of Michigan System-University of Michigan System | Laboratory of Growth Regulators [Univ Palacký] (LGR) ; Faculty of Science [Univ Palacký] ; Palacky University Olomouc-Palacky University Olomouc-Institute of Experimental Botany of the Czech Academy of Sciences (IEB / CAS) ; Czech Academy of Sciences [Prague] (CAS)-Czech Academy of Sciences [Prague] (CAS) | Czech Academy of Sciences [Prague] (CAS) | MICrobiologie de l'ALImentation au Service de la Santé (MICALIS) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech
Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N(6)-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N'-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N'-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity.
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