Mechanism of 8-Amino-7-oxononanoate Synthase:  Spectroscopic, Kinetic, and Crystallographic Studies^,

Abstract

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 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.

Histidine 282 in 5-Aminolevulinate Synthase Affects Substrate Binding and Catalysis

“…In both the glycine and succinyl-CoA soaked R. capsulatus ALAS crystals, a 15° rotation of the pyridine ring around the C5-C5A bonds occurs such that the O3 and C4A atoms move away from the catalytic lysine (10). Upon the binding of product in AONS, a similar rotation of the pyridine ring occurs along with subtle rearrangement of the active site hydrogen bond system (13). In R. capsulatus ALAS, the movement of the O3 is tracked by the residue equivalent to murine ALAS H282 (10), indicating that this residue is probably involved in coordinating the movement of the pyridine ring with the reorganization of the hydrogen bond system occurring upon substrate binding.…”

“…No studies have examined the role of this residue in any α-oxoamine synthase family member, although based on structural data alone it has been suggested that it may function as an acid catalyst during transaldimination (12,13), play a key role in positioning the PLP aromatic ring (11), or influence the pK a of the imine nitrogen (13).…”

Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters

“…1a) 7 . The x-ray structures and catalytic mechanisms of the E. coli AONS, DANS, DTBS and BS enzymes have been reported [8][9][10][11][12][13] .…”

“…The thioester is condensed with L-alanine in an AONScatalysed reaction to begin the late stages of biotin biosynthesis 8,13 . Labelling studies using 13 C acetate provided insight into the origin of the carbon atoms of pimeloyl-CoA and biotin in E. coli 15,16 .…”

Protein Assembly Line Components in Prodigiosin Biosynthesis: Characterization of PigA,G,H,I,J

“…Additional binding of PLP to the SerT domain of PigH could be shown by addition of a solution of pyridoxal phosphate to PigH. The absorption maximum of 388 nm, characteristic of the free aldehyde form of PLP, was shifted to 414 nm, typical of an aldimine linkage of PLP to an active-site lysine 8 ( Figure S3) in a 1:1 ratio. Thus, all three domains of PigH undergo post-translational modifications 9 in preparation for their assembly-line functions.…”