title: Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level.
abstract: Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg(2+) particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.
authors.affiliation: Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Marcuello
authors.forename: Sonia
authors.initials: S
authors.lastname: Arilla-Luna
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
authors.forename: Anabel
authors.initials: A
authors.lastname: Lostao
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D003352:Corynebacterium / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D000244:Adenosine Diphosphate; D005486:Flavin Mononucleotide; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase