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  • 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.

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.

Biochimica et biophysica acta (2013-01-08)
Carlos Marcuello, Sonia Arilla-Luna, Milagros Medina, Anabel Lostao
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.

MATERIALS
Product Number
Brand
Product Description

Riboflavin sodium phosphate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Riboflavin 5′-monophosphate sodium salt hydrate, suitable for electrophoresis, suitable for acrylamide photopolymerization, ≥70%, powder
Sigma-Aldrich
Riboflavin 5′-monophosphate sodium salt hydrate, 73-79% (fluorimetric)
Sigma-Aldrich
Riboflavin 5′-monophosphate sodium salt hydrate, synthetic, ≥70% (HPLC)
Sigma-Aldrich
Riboflavin Sodium Phosphate Hydrate, tested according to Ph. Eur.