Improving Catalytic Activity and Reversing Enantio-Specificity of ω-Transaminase by Semi-Rational Engineering en Route to Chiral Bulky β-Amino Esters

The application of wild-type omega-transaminase was limited by steric hindrance towards bulky substrates, therefore improvement of the catalytic efficiency and stereoselectivity toward substrates with two bulky substituent adjacent to the carbonyl is of general interest. In this study, according to the double substrate binding pocket theory, a (S)-selective omega-transaminase from the Burkholderia vietnamiensis G4, which showed puny catalytic activity toward the beta-keto esters with small steric hindrance, was engineered to accept bulky beta-keto esters, which were not accessible by any wild-type enzyme. A few desired variants were obtained that exhibited activity toward bulky beta-keto esters. Furthermore, a substrate-dependent shift in enantio-preference of HBV variant towards beta-keto esters with linear or branched aliphatic substituents was observed. The best variant was applied to the asymmetric synthesis of aliphatic beta-amino acids at semi-preparative scale with high yield and enantioselectivity. This study will improve the general understanding and inspire further engineering work for reversing enantio-specificity of omega-transaminases.

Automated summary

The study successfully engineered an omega-transaminase with (S)-selectivity to accept bulky substrates, resulting in several variants that showed activity towards bulky substrates, with the best variant achieving high yield and enantioselectivity in the asymmetric synthesis at a semi-preparative scale. This work will enhance the general understanding of omega-transaminases and inspire further engineering efforts to reverse enantio-specificity.