title: Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study.
abstract: 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.
authors.affiliation: Laboratoire de Biologie Cellulaire, INRA, Route de Saint-Cyr, F-78026 Versailles Cedex, France. kopecny_david@yahoo.co.uk
authors.forename: David
authors.initials: D
authors.lastname: Kopecný
authors.forename: Pierre
authors.initials: P
authors.lastname: Briozzo
authors.forename: Hana
authors.initials: H
authors.lastname: Popelková
authors.forename: Marek
authors.initials: M
authors.lastname: Sebela
authors.forename: Radka
authors.initials: R
authors.lastname: Koncitíková
authors.forename: Lukás
authors.initials: L
authors.lastname: Spíchal
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Nisler
authors.forename: Catherine
authors.initials: C
authors.lastname: Madzak
authors.forename: Ivo
authors.initials: I
authors.lastname: Frébort
authors.forename: Michel
authors.initials: M
authors.lastname: Laloue
authors.forename: Nicole
authors.initials: N
authors.lastname: Houba-Hérin
mesh_terms: D001483:Base Sequence; D018360:Crystallography, X-Ray; D003583:Cytokinins / Q000737:chemistry / Q000494:pharmacology*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics; D010802:Phylogeny; D010944:Plants / Q000235:genetics
chemical_list: D003583:Cytokinins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D010088:Oxidoreductases; C019984:cytokinin oxidase