An (R)-Selective Transaminase From Thermomyces stellatus

Stabilizing the Tetrameric Form

Journal article (2020)

Authors

C.M. Heckmann University of Nottingham

Louise Gourlay University of Milan

Beatriz Dominguez Johnson Matthey Technology Center

Francesca Paradisi University of Bern, University of Nottingham

Affiliation

External organisation

Biocatalysis Crystal structure Enzyme engineering Amino transferase Chiral amine Enzyme characterization Quaternary structure Thermostability

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Published Date

2020

Language

English

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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.