Structural and biochemical characterization of the dual substrate recognition of the (R)‐selective amine transaminase from Aspergillus fumigatus

Abstract

Chiral amines are important precursors for the pharmaceutical and finechemical industries. Because of this, the demand for enantiopure amines is currently increasing. Amine transaminases can produce a large spectrum of chiral amines in the (R)-or (S)-configuration, depending on their substrate scope and stereo-preference, by converting a prochiral ketone into the chiral amine while using alanine as the amine donor producing pyruvate as an a-keto acid product. In order to guide the protein engineering of transaminases to improve substrate specificity and enantioselectivity, we carried out a crystal structure analysis at 1.6A resolution of the (R)-amine transaminase from Aspergillus fumigatus with the bound inhibitor gabaculine. This revealed that Arg126 has an important role in the dual substrate recognition of this enzyme because mutating this residue to alanine reduced substantially the ability of the enzyme to use pyruvate as an amino acceptor. DatabaseCoordinates and structure factors have been deposited with the Protein Data Bank under accession code 4UUG.

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).…”

“…Up to now, several structures of S -ATAs9101112 and R -ATAs13141516 have been determined, providing some information about the dual substrate recognition30 of ATAs, which is a unique ability of transaminases to accept both the hydrophilic and hydrophobic substrates in the same active site. In the S -ATAs, a “flipping” arginine residue in a loop near the active site was proposed to play a key role in dual substrate recognition by moving the guanidino group in the active site to interact with the carboxylate of substrates or moving out of the active site to provide a hydrophobic environment9.…”

“…In the S -ATAs, a “flipping” arginine residue in a loop near the active site was proposed to play a key role in dual substrate recognition by moving the guanidino group in the active site to interact with the carboxylate of substrates or moving out of the active site to provide a hydrophobic environment9. Recently, R -ATAs from Aspergillus terreus , Nectria haematococca and Aspergillus fumigatus were reported and an arginine residue near the active site was assumed to possess the similar function, binding carboxylated groups, as that in the S -ATAs141516.…”

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

“…Interestingly, a flexible loop (e.g. aa K125-I135 in AT-ωTA), which is present in ( R )-ATAs13151735 as part of the active site (the loop of chain B as part of the active site of chain A and vice versa containing an arginine residue, which is discussed to play a role in dual substrate recognition of ( R )-ATAs131535) is significantly shorter in Cpu TA1 (aa E125-G128) and does not contain any positively charged amino acid. Thus, the loop is located further away from the PLP binding site creating more space in the active site (Fig.…”

“…6F). This loop changes its conformation from a closed to an open form upon binding of the inhibitor gabaculin in the ( R )-ATA of A. fumigatus 35 thereby moving R126 out of the active site, and due to high B-factors of the amino acid residues the loop is also expected to be flexible in AT-ωTA of A. terreus 15. Moreover, an altered conformation of the respective loop in ATA-117-Rd11 variant compared to its wildtype might be one of the reasons for its ability to accommodate the pro-sitagliptin ketone13.…”