AUTHOR=Heckmann Christian M. , Gourlay Louise J. , Dominguez Beatriz , Paradisi Francesca 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.00707

DOI=10.3389/fbioe.2020.00707

ISSN=2296-4185

ABSTRACT=<p>The identification and 3D structural characterization of a homolog of the (<italic>R</italic>)-selective transaminase (RTA) from <italic>Aspergillus terreus</italic> (<italic>At</italic>RTA), from the thermotolerant fungus <italic>Thermomyces stellatus</italic> (<italic>Ts</italic>RTA) is here reported. The thermostability of <italic>Ts</italic>RTA (40% retained activity after 7 days at 40°C) was initially attributed to its tetrameric form in solution, however subsequent studies of <italic>At</italic>RTA 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 <italic>Ts</italic>RTA_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. <italic>Ts</italic>RTA accepts the common amine donors (<italic>R</italic>)-methylbenzylamine, isopropylamine, and <sc>d</sc>-alanine as well as aromatic and aliphatic ketones and aldehydes.</p>