Application of reductive amination by heterologously expressed Thermomicrobium roseum L-alanine dehydrogenase to synthesize L-alanine derivatives
Küçük Resim Yok
Tarih
2023
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Elsevier Science Inc
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
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.
Açıklama
Anahtar Kelimeler
Unnatural Amino Acids, Medicinal Chemistry, Alanine Dehydrogenase, Thermomicrobium Roseum, Reductive Amination Mechanism, & Alpha;-Keto Acids, Molecular Modeling
Kaynak
Enzyme and Microbial Technology
WoS Q Değeri
N/A
Scopus Q Değeri
Q2
Cilt
169
