Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor.
Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N(6)-(buta-2,3-dienyl)adenine (HA-8) and N(6)-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 A resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.
Kopecný
David
D
Laboratoire de Biologie Cellulaire, INRA, Route de Saint-Cyr, F-78026 Versailles Cedex, France.
Sebela
Marek
M
Briozzo
Pierre
P
Spíchal
Lukás
L
Houba-Hérin
Nicole
N
Masek
Vlastimil
V
Joly
Nathalie
N
Madzak
Catherine
C
Anzenbacher
Pavel
P
Laloue
Michel
M
D001665:Binding Sites; D003583:Cytokinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014961:X-Ray Diffraction; D003313:Zea mays / Q000201:enzymology
D003583:Cytokinins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C019984:cytokinin oxidase