Figure 1.
Detailed reaction mechanism of transaminases.
Table 1.
Data collection and refinement statistics.
Figure 2.
A: Overview of the AT-ωTA dimer (chain A in green, chain B in grey), conserved regions are indicated in yellow, B: overview of the AT-ωTA dimer with the binding pockets indicated as orange spheres, C: small binding pocket amino acids (blue), D: large binding pocket amino acids (orange), E: PLP binding amino acids (green). The figures were prepared using the program PyMOL.
Figure 3.
Zoom into the loop Thr121-Val136 region of chain B in the structural alignment of the AT-ωTA structure (magenta) with other fold class IV transaminase structures (dark grey).
For PDB-IDs see Figure S2 in File S1. The figure was prepared using the program PyMOL.
Figure 4.
Docking of various substrate intermediates into AT-ωTA.
A: Pro-(R)- and (S)-acetophenone pyridoxal phosphate intermediates docked into the active site of AT-ωTA. Green: amino acids of the active site (chain A) and PLP bound to K180, blue: amino acids of the active site (chain B), purple: pro-(R)-acetophenone pyridoxal phosphate intermediate, turquoise: pro-(S)-acetophenone pyridoxal phosphate intermediate. B: Acetophenone pyridoxal phosphate intermediate (purple), propiophenone pyridoxal phosphate intermediate (blue), butyrophenone pyridoxal phosphate intermediate (turquoise) docked into the active site of AT-ωTA compared to PLP bound to K180 in the structure of AT-ωTA (green). Green: amino acids of the active site (chain A), blue: amino acids of the active site (chain B). The figures were prepared using the program PyMOL.
Figure 5.
Comparison of fold IV and fold I transaminases.
A: Position of lysine relative to PLP in fold IV transaminases: in AT-ωTA (green), BCAT from human (1KT8, blue) or E. coli (1IYE, turquoise) and D-ATA from Bacillus sp. YM-1 (3DAA, brown). Ligands: blue: L-Ile-aldimine bound in human BCAT, turquoise: L-Glu-aldimine bound in BCAT from E. coli, brown: D-Ala-aldimine bound in D-ATA, green: L-Glu-aldimine bound in AT-ωTA, purple: docked acetophenone-aldimine in AT-ωTA. B: Position of lysine relative to PLP in fold I (S)-ω-transaminases: in PD-ωTA from Paracoccus denitrificans (4GRX, light green), PA-ωTA from Pseudomonas aeruginosa (4B98, turquoise) and several (S)-TAs identified from the Pdb by Steffen-Munsberg: from Pseudomonas putida (3A8U, pink), from Mesorhizobium loti (3GJU, yellow) and from Silicobacter pomeroyi (3HMU, brownish), in PD-ωTA the substrate, 5-aminopentanoate, is depicted in light green. The figures were prepared using the program PyMOL.
Table 2.
Localisation of the large and small binding pocket relative to PO4 and O3’ of PLP and binding of the substrates’ substituents in different amino acid and amine transaminases (upper part: fold I aminotransferases, lower part: fold IV aminotransferases).
Figure 6.
Schematic drawing of the localisation of the large and small binding pocket in AT-ωTA relative to PO4 and O3’ of PLP and the binding of the substrates’ substituents.
Figure 7.
Relative activities calculated from the increase of acetophenone at 300-ωTA and mutants thereof (0.1 mM PLP, 5 mM (R)-α-methylbenzylamine, 5 mM pyruvate or 5 mM butanal, in 50 mM KPi, pH 7.5, 0.25 mg/mL of total lysate protein) at 25°C.
The relative activities are referred to either the wild-type activity with pyruvate (0.8 U/mg lysate, dark grey bars) or butanal (0.1 U/mg lysate, light grey bars).