Functional and structural insights into a thermostable (S)-selective amine transaminase and its improved substrate scope by protein engineering

TL;DR

Scientists improved a heat-resistant enzyme to work with a wider range of substances, making it more useful for industrial applications.

Contribution

A thermostable amine transaminase was engineered to accept bulky aromatic substrates through site-directed mutagenesis.

Findings

Sbv333-ATA is stable at high temperatures and in various organic solvents.

The mutant W89A can process bulky diaromatic amines previously rejected by the native enzyme.

High-resolution structures of the enzyme and its mutants revealed active site details.

Abstract

A (S)-selective amine transaminase from a Streptomyces strain, Sbv333-ATA, is a biocatalyst showing both high thermostability with a melting temperature of 85 °C and broad substrate specificity for the amino acceptor. This enzyme was further characterized both biochemically and structurally. The Sbv333-ATA is stable in the presence of up to 20% (v/v) of the water-miscible cosolvents methanol, ethanol, acetonitrile, and dimethyl sulfoxide, and in biphasic systems with petroleum ether, toluene, and ethyl acetate as an organic phase. The enzyme showed also a good activity toward different amino donors, such as (S)-methylbenzylamine and 2-phenylethylamine, aliphatic mono- and di-amines, like propylamine and cadaverine, and selected amino acids. However, more sterically hindered aromatic amines were not accepted. Based on the knowledge of the three-dimensional structures obtained, a rational approach to site specific mutagenesis was carried out to broaden the substrate specificity of Sbv333-ATA. The mutant W89A showed the highest activity toward bulky amines as substrates, such as the diaromatic compound 1,2-diphenylethylamine. The 3D structures of the holo and inhibitor gabaculine bound forms of native Sbv333-ATA, and holo W89A and F61C mutants were determined at high resolutions of 1.49, 1.24, and 1.31 (both mutants) Å, respectively. These structures were important for revealing further details of the active site binding pockets of the Sbv333-ATA and its mechanism.

• Sbv333-ATA is a highly thermostable transaminase with a broad substrate scope.

• Sbv333-ATA remains active in various organic cosolvents and biphasic systems.

• Mutant W89A expands substrate range to accept bulky diaromatic amines.

The online version contains supplementary material available at 10.1007/s00253-025-13536-9.

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