title: Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site.
abstract: Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins.
authors.affiliation: Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: David
authors.identifier: http://orcid.org/0000-0002-4309-4284
authors.initials: D
authors.lastname: Kopečný
authors.affiliation: Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: Radka
authors.initials: R
authors.lastname: Končitíková
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Hana
authors.initials: H
authors.lastname: Popelka
authors.affiliation: Institut Jean-Pierre Bourgin, UMR1318 INRA, AgroParisTech, Versailles, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Briozzo
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CNRS-CEA-Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
authors.forename: Armelle
authors.initials: A
authors.lastname: Vigouroux
authors.affiliation: Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Kopečná
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Zalabák
authors.affiliation: Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: Marek
authors.initials: M
authors.lastname: Šebela
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Skopalová
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.
authors.forename: Ivo
authors.initials: I
authors.lastname: Frébort
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CNRS-CEA-Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
authors.forename: Solange
authors.initials: S
authors.lastname: Moréra
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003583:Cytokinins / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology
chemical_list: D003583:Cytokinins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C019984:cytokinin oxidase
keywords: crystal structure; cytokinin oxidase/dehydrogenase; flavoprotein; maize; plant hormone; site-directed mutagenesis