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

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

doi:10.3389/fbioe.2020.00707

Christian M Heckmann, Beatriz Dominguez + Show 2 more

Open Access

https://doi.org/10.3389/fbioe.2020.00707

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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.

Highlights

A green chemistry approach to minimize the environmental impact of synthetic processes entails the use of biocatalysts, as they often offer unmatched regio, enantio, and chemo-selectivity (Truppo, 2017)
thermotolerant fungus Thermomyces stellatus (TsRTA) was readily expressed at 25◦C and, following Ni-immobilized metal affinity chromatography (IMAC), the enzyme was obtained in high yields (800 mgenzyme/Lculture) and was judged to be pure by SDS-PAGE (Figure S2)
Upon comparisons made between the crystal structure of TsRTA and

Summary

Introduction

A green chemistry approach to minimize the environmental impact of synthetic processes entails the use of biocatalysts, as they often offer unmatched regio-, enantio-, and chemo-selectivity (Truppo, 2017). At the forefront of industrial applications, are transaminases (TAs) (Fuchs et al, 2015; Kelly et al, 2018), pyridoxal-5′-phosphate (PLP) dependent enzymes catalyzing the shuttling of an amino group between an amine and a carbonyl group, producing chiral primary amines, a highly desired reaction in industry (Constable et al, 2007; Savile et al, 2010). Both (S)-selective transaminases (STAs) and (R)-selective transaminases (RTAs), belonging to separate fold types within the family of PLP-dependent enzymes, are known. The overall number of known RTAs (and their solved structures) is still significantly smaller compared to STAs (Guo and Berglund, 2017; Slabu et al, 2017)

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