Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site. (26th November 2015)

• Record Type: Journal Article
• Authors: Kopečný, David; Končitíková, Radka; Popelka, Hana; Briozzo, Pierre; Vigouroux, Armelle; Kopečná, Martina; Zalabák, David; Šebela, Marek; Skopalová, Jana; Frébort, Ivo; Moréra, Solange
• Is Part Of: FEBS journal. Volume 283:Number 2(2016)
• Page Start: 361
• Page End: 377
• Publication Date: 2015-11-26
• Subjects: crystal structure -- cytokinin oxidase/dehydrogenase -- flavoprotein -- maize -- plant hormone -- site‐directed mutagenesis; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.13581
• Languages: English
• ISSNs: 1742-464X

Abstract

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‐ N 1‐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 N 6‐(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. Abstract : The role of seven active site residues of the cytokinin oxidase/dehydrogenase CKO1 from maize (ZmCKO1) was studied combining site‐directed mutagenesis with kinetics and X‐ray crystallography. New structures of ZmCKO2 and ZmCKO4a were solved. We found that D169 is important for catalysis, H105 covalently linked to FAD maintains the enzyme's structural integrity and E381 restricts binding of cytokinin ribosides and glucosides.