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

Huri Dedeakayoğulları, Jarkko Valjakka, Ossi Turunen, Berin Yilmazer, Ğarip Demir, Janne Jänis, Barış Binay

    Research output: Contribution to journalArticleScientificpeer-review


    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.

    Original languageEnglish
    Article number110265
    Number of pages10
    Publication statusPublished - Sept 2023
    Publication typeA1 Journal article-refereed


    • Alanine dehydrogenase
    • Medicinal chemistry
    • Molecular modeling
    • Reductive amination mechanism
    • Thermomicrobium roseum
    • Unnatural amino acids
    • α-keto acids

    Publication forum classification

    • Publication forum level 1

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering
    • Biochemistry
    • Applied Microbiology and Biotechnology


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