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 8a-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.

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567‐A

“…The structure of LuCKX1.1 exhibits a two‐domain topology consisting of a flavin adenine dinucleotide (FAD)‐binding domain and a substrate‐binding domain (Fig. ), which is similar to the known structures of AtCKX7, ZmCKX1, ZmCKX2, ZmCKX4a and ZmCKX4b (Bae et al , ; Kopecny et al , ; Malito et al , ). LuCKX1.1 contains the FAD co‐factor covalently linked to histidine‐104 (His104) via the 8‐methyl group of the flavin ring in LuCKX1.1.…”

“…The LuCKX1.1 crystal structure is similar to that known for other CKX enzymes with a two‐domain topology (Bae et al , ; Kopecny et al , ; Malito et al , ). A 72‐amino‐acid region sufficient for Y2H interaction with AvrL567 (Fig.…”

“…The other domain (residues 244–477) contains an antiparallel β‐sheet surrounded by α‐helices. The corresponding domain in the structures of ZmCKX1 and ZmCKX2 complexed with different cytokinins is involved in substrate binding (Kopecny et al , ; Malito et al , ). The residues important for substrate binding identified in the active sites of ZmCKX1 and ZmCKX2 are largely conserved in LuCKX1.1, including aspartic acid‐168 (Asp168), glutamic acid‐281 (Glu281), valine‐365 (Val365), glutamic acid‐369 (Glu369), tryptophan‐384 (Trp384), proline‐413 (Pro413), leucine‐446 (Leu446) and leucine‐483 (Leu483).…”

“…), further suggesting the involvement of surface 1 in this interaction. A recent study on the structures of several ZmCKX isoforms (Kopecny et al , ) has suggested that a region corresponding to surface 1 in ZmCKX could potentially be involved in the determination of substrate specificity and activity. Thus, the binding of Avr567‐A to surface 1 could influence LuCKX1.1 enzymatic activity by altering substrate access to the active site.…”

“…The regions corresponding to surface 1 in other known CKX structures vary in both sequence and structural conformation. The structures of maize CKXs show that only ZmCKX1 adopts a well‐ordered conformation in the region corresponding to surface 1 with two helices (Kopecny et al , ; Malito et al , ). In Arabidopsis AtCKX7, this region corresponds to two short helices with connecting loops (Bae et al , ) and, in flax LuCKX1.1, it largely comprises a loop containing a short helix.…”