Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Abstract

Background: ␤-Transaminases are promising biocatalysts for the synthesis of ␤-amino acids. Results: The first three-dimensional structures were obtained of a native ␤-transaminase and complexes with a keto acid and two covalently bound ␤-amino acids. Conclusion: Dual functionality of the carboxylate-and side chain-binding pockets allows binding of ␤-and ␣-amino acids. Significance: These structures may facilitate the development of improved ␤-amino acid biocatalysts.

Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax paradoxus

“…These data suggest that VpAT, just like MesAT and PoGSAM, is a fold type I aminotransferase, and furthermore, that VpAT belongs to subgroup II transaminases, which is based on an enzyme's substrate specificity (27,48,68). VpAT has 19% sequence identity to a transaminase from Alcaligenes denitrificans that has been reported to have activity toward aliphatic ␤-amino acids (AdbpAT) (AAP92672.1) (21).…”

“…The monomer consists of 11 ␣-helices of at least 2 turns and 13 ␤-strands that form a central 7-stranded mixed ␤-sheet in the PLP-binding domain and two 3-stranded antiparallel ␤-sheets in the NC domain. The overall structure of the enzyme is similar to that of aspartate aminotransferase, the archetype of a fold type I aminotransferase (z-score, 22; RMSD, 4.3 Å for 308 C-␣ atoms; 17% sequence identity; PDB code 1BKG) (70,71), but is most similar to that of MesAT (z-score, 60; RMSD, 1.1 Å for 429 C-␣ atoms; 50% sequence identity; PDB code 2YKU) (48).…”

“…The structure of VpAT with AOA ( Fig. 3A and B) shows that the amino group of the inhibitor covalently binds to the C-4A atom of the PLP cofactor, as also described for aspartate aminotransferase (73) and recently for MesAT (48). The ether-oxygen atom (O1) of AOA is positioned close (3.0 Å) to the ε-amino group of residue K267.…”

“…The 1.3-kb PCR product was cloned using the NdeI and XhoI sites of the pET28bϩ plasmid. The MesAT I56V/A312S/M414F triple mutant was expressed and purified as previously reported for the MesAT wild type (WT) (48).…”

“…Indexing and integration of reflections was done using XDS (49), and scaling and merging of the data were achieved using SCALA (50) from the CCP4 software suite (51). For molecular replacement, the Phaser program (52) was used with MesAT (Protein Data Bank [PDB] code 2YKU [48]) as the input model. The resulting model structure was subjected to successive rounds of automatic model building using ARP/wARP (53) at 1.7-Å resolution, followed by manual model building and manipulation in Coot (54).…”