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497 a.a.
 N(6)-dimethylallyladenine |
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acceptor |
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H(2)O |
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 adenine |
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 3-methylbut-2-enal |
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reduced acceptor |
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 FAD |
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Key reference
DOI no: 10.1016/j.jmb.2008.05.044 J Mol Biol 380:886-899 (2008) PubMed id: 18571199
Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor. D.Kopecný, M.Sebela, P.Briozzo, L.Spíchal, N.Houba-Hérin, V.Masek, N.Joly, C.Madzak, P.Anzenbacher, M.Laloue. ABSTRACT
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.
Selected figure(s)
Figure 4.
Fig. 4. Crystal structure of ZmCKO1 inactivated by allenic cytokinins. (a) Overall surface of the enzyme. β-Strands are indicated in cyan and α-helices in light blue. FAD cofactor (yellow), bound HA-1 inhibitor (green) and N-acetyl-glucosamines (orange) are shown in space-filling CPK representation with nitrogen atoms in blue and oxygen atoms in red. The five glycosylated Asn sites are numbered. (b and c) Binding of HA-1 (carbon atoms in green) and HA-8 (carbon atoms in black) in their 2F[o] − F[c] maps; contoured at 2σ. The new covalent bond between the C3 atom of the inhibitor side chain and the C4a atom of FAD is shown in brown. (d) Hydrogen-bonding interactions of the inhibitor HA-1 at the active site of ZmCKO1. (e) Detail of the covalent bond between HA-1 inhibitor and FAD cofactor.Figure 6.
Fig. 6. Mechanism of ZmCKO1 inactivation by allenic substrate analogs. In the first step (reductive half-reaction), the enzyme reacts with HA-1 or HA-8 and, as a result, the FAD cofactor is reduced and the corresponding allenic imine intermediate released. Then the allenic intermediate imine undergoes hydrolysis to form the corresponding allenic aldehyde and adenine followed by cofactor reoxidation (route A) or the strongly electrophilic C3 atom of the allenic imine intermediate attacks the C4a atom of the reduced cofactor and a covalent modification of the cofactor occurs (HA–FAD adduct, route B).The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 380, 886-899) copyright 2008. Figures were selected by an automated process.
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