Exploring the Stability and Substrate Profile of Transaminase from Silicibacter pomeroyi with Ancestral Sequence Reconstruction.

Abstract

Amine transaminases (ATAs), belonging to the class III transaminases within the superfamily of pyridoxal-5'-phosphate (PLP)-dependent enzymes, catalyze transamination reactions between amino donors and amino acceptors. These enzymes are particularly appealing for their role in the stereospecific synthesis of chiral amines. However, the stability of most ATAs is not satisfying, limiting their suitability for industrial applications. Among them, the amine transaminase from Silicibacter pomeroyi (Sp-ATA) has drawn attention due to its high activity and broad substrate scope under mild conditions and high pH. Nevertheless, maintaining the activity at higher temperatures presents a challenge. Previous research to enhance enzyme function through directed evolution has shown promise, yet predicting the cooperative effects of individual stabilizing mutations remains challenging. Computational approaches have been explored but often rely on structural information and are resource-intensive. An alternative strategy is ancestral sequence reconstruction (ASR), which is based on gene sequences to create a more or less artificial phylogenetic tree. This study aims to leverage ASR techniques to explore the thermostability, solvent tolerance, and substrate profile of Sp-ATA, to find highly stable transaminases. By using Sp-ATA as a template and incorporating insights from ancestral sequences, this strategy offers a promising approach for developing robust biocatalysts.