Sbv333-ATA from Streptomyces sp.: Discovery, Functional Characterization, and Engineering

Aminotransferases (ATAs), PLP-dependent enzymes, catalyze the formation of chiral amines, making them a valuable tool for different industrial applications. Using functional screening and genome mining, a putative transaminase gene was identified in Streptomyces sp. BV333 showing sequence similarity to thermostable ATAs [1]. The corresponding enzyme, Sbv333-ATA, was successfully expressed in E. coli and exhibited exceptional thermostability (Tm = 85 °C) and broad substrate specificity. Notably, Sbv333-ATA efficiently transaminated β- ketoesters, a rare feature among known ATAs [2]. Recently, new studies have been performed to assess enzyme stability in the presence of organic (co)solvents. Sbv333-ATA proved to be stable in up to 20% (v/v) water-miscible co-solvents, including methanol, ethanol, acetonitrile, and dimethyl sulfoxide, as well as in biphasic systems with petroleum ether, toluene, and ethyl acetate as the organic phase. Additionally, a panel of amine donors was tested to further investigate the substrate scope. The enzyme was also successfully crystallized, and high-resolution structures of both its native form and its complex with the inhibitor gabaculine were solved. Structural analysis revealed key features of the active site architecture, providing valuable insights into the amino acids to target for rational mutagenesis to enhance enzyme performance. This approach enabled the design of improved variants with expanded active site, allowing for more efficient interactions with bulky substrates and ultimately increasing the enzyme’s catalytic potential [3]. [1] E. E. Ferrandi, A. Previdi, I. Bassanini, S. Riva, X. Peng, D. Monti, Appl. Microbiol. Biotechnol., 2017, 101, 4963-4979. [2] E. E. Ferrandi, J. Spasic, L. Djokic, Y. Vainshtein, R. Senthamaraikannan et al., Catalysts, 2021, 11, 919. [3] S. Patti, S. A. De Rose, M. N. Isupov, I. Magrini Alunno, S. Riva, E. E. Ferrandi, J. A. Littlechild, D. Monti (manuscript in preparation).