Application of reductive amination by heterologously expressed Thermomicrobium roseum L-alanine dehydrogenase to synthesize L-alanine derivatives

Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, a-ketobutyrate, a-ketovalerate and a-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic k(cat) values were significantly affected by the side chain increase. In contrast, K-M values associated with the derivatives of L-alanine (L-a-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/a-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.

Automated summary

Unnatural amino acids, with amino and carboxylic acid functional groups and variable side chains, are important building blocks in modern medicinal chemistry. They can be synthesized from natural amino acids or through the use of enzymes, leading to novel molecules used in pharmaceutical production. This study focused on the reductive amination activity of the Thermomicrobium roseum alanine dehydrogenase enzyme, examining its potential with different substrates and the effects of metal ions on enzymatic activity. The enzyme showed promising reductive activity and may have applications in the synthesis of pharmaceutically relevant amino acids.