RESUMEN
Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism. The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained. Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s-1mM-1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against α-ketobutyrate (34.0 s-1mM-1) and α-ketovalerate (2.7 s-1mM-1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s-1mM-1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.
RESUMEN
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, α-ketobutyrate, α-ketovalerate and α-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 kcat values were significantly affected by the side chain increase. In contrast, KM values associated with the derivatives of L-alanine (L-α-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/α-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.