Crystallographic characterization of the (R)-selective amine transaminase fromAspergillus fumigatus

Abstract

The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years. The X-ray crystal structure of the (R)-selective amine transaminase from the fungus Aspergillus fumigatus was solved by S-SAD phasing to 1.84 Å resolution. The refined structure at 1.27 Å resolution provides detailed knowledge about the molecular basis of substrate recognition and conversion to facilitate protein-engineering approaches. The protein forms a homodimer and belongs to fold class IV of the pyridoxal-5'-phosphate-dependent enzymes. Both subunits contribute residues to form two active sites. The structure of the holoenzyme shows the catalytically important cofactor pyridoxal-5'-phosphate bound as an internal aldimine with the catalytically responsible amino-acid residue Lys179, as well as in its free form. A long N-terminal helix is an important feature for the stability of this fungal (R)-selective amine transaminase, but is missing in branched-chain amino-acid aminotransferases and D-amino-acid aminotransferases.

A new target region for changing the substrate specificity of amine transaminases

“…ATAs transfer an amino group of a chiral amine compound to a ketone compound using the cofactor pyridoxal 5’-phosphate (PLP) (Supplementary Fig. 1a) with a high turnover rate, stable catalytic activity, broad substrate specificity and excellent stereoselectivity7, which is achieved by a proposed large-binding pocket (L pocket) and small-binding pocket (S pocket) in the substrate-binding site8910111213141516 (Supplementary Fig. 1b).…”

Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold

“…Compared to the structure of ( R )-ATAs, e.g. of A. terreus and A. fumigatus, the active site of Cpu TA1 is further slightly opened up as the interdomain loop E138-Q144 is located further away from the PLP (corresponding to loop P144-A150 containing I146 and V148 which were predicted to be part of the active site of the ( R )-ATA of A. fumigatus 18. In contrast, the respective loop in D-ATAs is located significantly further away, which is in correspondence with the prediction that the positions of the small and the large binding pocket in relation to the O3′ and the phosphate group of PLP are complementary in ( R )-ATA and D-ATAs.…”

“…PDB-code: 4CE5 from Aspergillus terreus 15, Z-score: 29.0, rmsd 2.2 Å, seq id: 22%). ( R )-selective amine transaminases contain an N-terminal α-helix spanning the initial 20 amino acids1518, which is not observed in any of the other fold IV subfamilies. This helix is also not present in Cpu TA1 (Fig.…”

“…D-ATAs: 1DAA from Bacillus sp YM-131, 4M0J from Burkholderia thailandensis E264; R-ATAs: 4CE5 from Aspergillus terreus 15, 4CHI from Aspergillus fumigatus 18, 4CMD from Nectria haematococca 17, 3WWH from Arthrobacter sp. KNK16813; BCATs: 1IYE from E. coli 32, 2EIY from Thermus thermophilus , 3HT5 from Mycobacterium tuberculosis 47, 3JZ6 from Mycobacterium smegmatis 48, 3UYY from Deionococcus radiodurans 49, 3U0G from Burkholderia pseudomallei , 4DQN from Streptococcus mutans 50, 2COI36 and 2HG851 from human; ADCLs: 1I2K from E. coli , 2Y4R from Pseudomonas aeruginosa 52, and a few putative aminotransferases: 2ZGI from Thermus thermophilus HB853, 3CEB from Haemophilus somnus , 3CSW from Thermotoga maritima , 3LUL from Legionella pneumophila , 3QQM from Mesorhizobium loti, 3SNO from Corynebacterium glutamicum , 4JXU from Sinorhizobium meliloti and 4TVI from Brucella abortus.…”

“…Although a lot of effort has already been made to improve and tailor transaminases according to industrial requirements by protein, substrate and reaction engineering89101112, there is still a clear need for further enhancement of known transaminases or the discovery of new, especially ( R )-selective transaminases (( R )-ATA) exhibiting improved properties. All ( R )-amine transaminases described in the literature so far are members of the fold type IV PLP-dependent enzyme family131415161718. This family includes d -amino acid aminotransferases, L-branched chain aminotransferases and 4-amino-4-deoxychorismate lyases (ADCL)19.…”