An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form

Christian M Heckmann; Louise J Gourlay; Beatriz Dominguez; Francesca Paradisi

doi:10.3389/fbioe.2020.00707

An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form

Front Bioeng Biotechnol. 2020 Jul 22:8:707. doi: 10.3389/fbioe.2020.00707. eCollection 2020.

Authors

Christian M Heckmann¹, Louise J Gourlay², Beatriz Dominguez³, Francesca Paradisi^{1

4}

Affiliations

¹ School of Chemistry, University of Nottingham, Nottingham, United Kingdom.
² Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
³ Johnson Matthey, Cambridge, United Kingdom.
⁴ Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.

Abstract

The identification and 3D structural characterization of a homolog of the (R)-selective transaminase (RTA) from Aspergillus terreus (AtRTA), from the thermotolerant fungus Thermomyces stellatus (TsRTA) is here reported. The thermostability of TsRTA (40% retained activity after 7 days at 40°C) was initially attributed to its tetrameric form in solution, however subsequent studies of AtRTA revealed it also exists predominantly as a tetramer yet, at 40°C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with TsRTA_G205C retaining almost full activity after incubation at 50°C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. TsRTA accepts the common amine donors (R)-methylbenzylamine, isopropylamine, and d-alanine as well as aromatic and aliphatic ketones and aldehydes.

Keywords: amino transferase; biocatalysis; chiral amine; crystal structure; enzyme characterization; enzyme engineering; quaternary structure; thermostability.