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

A ( S )-selective amine transaminase from a Streptomyces strain, Sbv333-ATA is a biocatalyst showing both high thermostability with a melting temperature of 85 ^o C and broad substrate specificity for the amino acceptor. This enzyme has been further characterised 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, dimethyl sulfoxide, and in biphasic systems with petroleum ether, toluene and ethyl acetate as an organic phase. The enzyme showed also a good activity towards different amino donors, such as ( S )-methylbenzylamine and 2-phenylethylamine, aliphatic mono- or di-amines like propylamine, cadaverine, and putrescine, and selected amino acids. However, more sterically hindered aromatic amines are not accepted. Based on the knowledge of the three-dimensional structures obtained a rational approach to site specific mutagenesis has been carried out to broaden the substrate specificity of Sbv333-ATA. A mutant W89A showed the highest activity towards bulky amines as substrates, such as the diaromatic compound 1,2-diphenylethylamine. The high-resolution structures of the holo and inhibitor gabaculine bound forms of native Sbv333-ATA, and holo W89A and F61C mutants have been determined at high resolutions of 1.49, 1.24 and 1.31 (both mutants) Å respectively. These structures have been important for revealing further details of the active site binding pockets of the Sbv333-ATA and its enzyme mechanism.