RESUMO
The quaternary structure of a fatty acid beta-oxidation multienzyme complex, catalyzing three sequential reactions, was investigated by X-ray crystallographic and small-angle X-ray solution scattering analyses. X-ray crystallography revealed an intermediate structure of the complex among the previously reported structures. However, the theoretical scattering curves calculated from the crystal structures remarkably disagree with the experimental profiles. Instead, an ensemble of the atomic models, which were all calculated by rigid-body optimization, reasonably explained the experimental data. These structures significantly differ from those in the crystals, but they maintain the substrate binding pocket at the domain boundary. Comparisons among these structures indicated that binding of 3-hydroxyhexadecanoyl-CoA or nicotinamide adenine dinucleotide induces domain rearrangements in the complex. The conformational changes suggest the structural events occurring during the chain reaction catalyzed by the multienzyme complex.
Assuntos
Modelos Moleculares , Complexos Multienzimáticos/química , Catálise , Cristalografia por Raios X , Dimerização , Ligantes , Proteína Mitocondrial Trifuncional , Modelos Químicos , Complexos Multienzimáticos/metabolismo , NAD/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Especificidade por SubstratoAssuntos
Complexos Multienzimáticos/química , Complexos Multienzimáticos/fisiologia , 3-Hidroxiacil-CoA Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/fisiologia , Acetil-CoA C-Aciltransferase/química , Acetil-CoA C-Aciltransferase/fisiologia , Animais , Bactérias/enzimologia , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/fisiologia , Humanos , Mitocôndrias/enzimologia , Proteína Mitocondrial Trifuncional , Oxirredução , Ligação Proteica , Especificidade por SubstratoRESUMO
The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid beta-oxidation cycle. The alpha2beta2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3'-phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3'-phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The alpha-helical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other beta-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.
