Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis.
Cytokinins form a diverse class of compounds that are essential for plant growth. Cytokinin dehydrogenase has a major role in the control of the levels of these plant hormones by catalysing their irreversible oxidation. The crystal structure of Zea mays cytokinin dehydrogenase displays the same two-domain topology of the flavoenzymes of the vanillyl-alcohol oxidase family but its active site cannot be related to that of any other family member. The X-ray analysis reveals a bipartite architecture of the catalytic centre, which consists of a funnel-shaped region on the protein surface and an internal cavity lined by the flavin ring. A pore with diameter of about 4A connects the two active-site regions. Snapshots of two critical steps along the reaction cycle were obtained through the structural analysis of the complexes with a slowly reacting substrate and the reaction product, which correspond to the states immediately before (Michaelis complex) and after (product complex) oxidation has taken place. The substrate displays a "plug-into-socket" binding mode that seals the catalytic site and precisely positions the carbon atom undergoing oxidation in close contact with the reactive locus of the flavin. A polarising H-bond between the substrate amine group and an Asp-Glu pair may facilitate oxidation. Substrate to product conversion results in small atomic movements, which lead to a planar conformation of the reaction product allowing double-bond conjugation. These features in the mechanism of amine recognition and oxidation differ from those observed in other flavin-dependent amine oxidases.
Malito
Enrico
E
Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy.
Coda
Alessandro
A
Bilyeu
Kristin D
KD
Fraaije
Marco W
MW
Mattevi
Andrea
A
D000225:Adenine / Q000031:analogs & derivatives; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003583:Cytokinins / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D003313:Zea mays / Q000201:enzymology*; D015026:Zeatin / Q000737:chemistry / Q000378:metabolism
D003583:Cytokinins; D010940:Plant Proteins; D005182:Flavin-Adenine Dinucleotide; D015026:Zeatin; D010088:Oxidoreductases; D000225:Adenine