Crystal structure of an (R)-selective ω-transaminase from Aspergillus terreus

Abstract

Chiral amines are important building blocks for the synthesis of pharmaceutical products, fine chemicals, and agrochemicals. ω-Transaminases are able to directly synthesize enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5'-phosphate (PLP) as cofactor. In nature, (S)-selective amine transaminases are more abundant than the (R)-selective enzymes, and therefore more information concerning their structures is available. Here, we present the crystal structure of an (R)-ω-transaminase from Aspergillus terreus determined by X-ray crystallography at a resolution of 1.6 Å. The structure of the protein is a homodimer that displays the typical class IV fold of PLP-dependent aminotransferases. The PLP-cofactor observed in the structure is present in two states (i) covalently bound to the active site lysine (the internal aldimine form) and (ii) as substrate/product adduct (the external aldimine form) and free lysine. Docking studies revealed that (R)-transaminases follow a dual binding mode, in which the large binding pocket can harbour the bulky substituent of the amine or ketone substrate and the α-carboxylate of pyruvate or amino acids, and the small binding pocket accommodates the smaller substituent.

Figure 1. Detailed reaction mechanism of transaminases.

Figure 2. Crystal structure of AT-ωTA.

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.

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).