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

摘要: 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 stereospecific manner to form 8(S)-amino-7-oxononanoate. This first committed step biotin biosynthesis. The mechanism Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. X-ray structure holoenzyme refined at resolution 1.7 A (R = 18.6%, R(free) 21. 2%) shows that plane imine bond internal aldimine deviates from pyridine plane. enzyme-product external complex 2.0 21.2%, 27.8%) rotation ring respect aldimine, together significant conformational change C-terminal domain subtle rearrangement active site hydrogen bonding. reaction, formation, rapid (k(1) 2 x 10(4) M(-)(1) s(-)(1)). Formation an D-alanine, not substrate, significantly slower 125 Binding D-alanine enhanced approximately 2-fold presence pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition preformed preceded distinct lag phase ( 30 ms) suggests binding causes transition complex. transition, inferred modeling require around Calpha-N complex, promotes abstraction Calpha proton Lys236. These results have combined detailed mechanistic pathway for catalysis may be applied other members alpha-oxoamine subfamily.