Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies

Biochemistry. 2000 Jan 25;39(3):516-28. doi: 10.1021/bi991620j.


8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 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 A (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 A (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 (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 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 ( approximately 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 Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha 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 alpha-oxoamine synthase subfamily.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / metabolism
  • Acyltransferases / chemistry*
  • Acyltransferases / metabolism*
  • Alanine / metabolism
  • Amino Acid Sequence
  • Bacillus / enzymology
  • Binding Sites
  • Crystallography, X-Ray
  • Escherichia coli / enzymology*
  • Hydrogen Bonding
  • Kinetics
  • Mass Spectrometry
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Spectrophotometry
  • Substrate Specificity


  • Acyl Coenzyme A
  • pimeloyl-coenzyme A
  • Acyltransferases
  • 8-amino-7-oxononanoate synthase
  • Alanine

Associated data

  • PDB/IDJ9