8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5‘-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of l-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 Å (R = 18.6%, R_free = 21.2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme−product external aldimine complex has been refined at a resolution of 2.0 Å (R = 21.2%, R_free = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, l-alanine external aldimine formation, is rapid (k₁ = 2 × 10⁴ M^-1 s^-1). Formation of an external aldimine with d-alanine, which is not a substrate, is significantly slower (k₁ = 125 M^-1 s^-1). Binding of d-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed l-alanine external aldimine complex and is preceded by a distinct lag phase (∼30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Cα−N bond of the external aldimine complex, promotes abstraction of the Cα proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the α-oxoamine synthase subfamily.