Functional and Structural Characterization of a Thermostable (S)-Selective Amine Transaminase with Enhanced Substrate Scope via Protein Engineering

Recently, our group identified in the genome of Streptomyces sp. BV333 an (S)-selective amine transaminase Sbv333-ATA which exhibits remarkable thermostability and broad substrate specificity toward various amino acceptors. In the present work, this interesting biocatalyst has been further studied from a functional point of view and structurally characterized. Functional characterization revealed its stability in up to 20% (v/v) of water- miscible co-solvents (methanol, ethanol, acetonitrile, dimethyl sulfoxide) and in biphasic systems with petroleum ether, toluene, or ethyl acetate. The enzyme demonstrated activity with diverse amino donors, including (S)- methylbenzylamine, 2-phenylethylamine, and aliphatic mono- or di-amines, but it could not process sterically hindered aromatic amines. High-resolution crystal structures were obtained for the native enzyme and complexes with substrates and the inhibitor gabaculine. Guided by these structural insights, rational mutagenesis was employed to enhance substrate specificity. Among tested variants, W89A demonstrated significantly improved activity with bulky substrates, paving the way for further optimization of Sbv333-ATA as a versatile biocatalyst.