High-Throughput Screening and Directed Evolution of Methionine Adenosyltransferase from Escherichia coli

S-adenosyl-L-methionine (SAM) is the active form of methionine, which participates in various metabolic reactions and plays a vital role. It is mainly used as a precursor by three key metabolic pathways: trans-methylation, trans-sulfuration, and trans-aminopropylation. Methionine adenosyltransferase (MAT) is the only enzyme to produce SAM from methionine and ATP. However, there is no efficient and accurate method for high-throughput detection of SAM, which is the major obstacles of directed evolution campaigns for MAT. Herein, we established a colorimetric method for directed evolution of MAT based on detecting SAM by using glycine oxidase and glycine/sarcosine N-methyltransferase enzyme. Screening of MAT libraries revealed variant I303V/Q22R with 2.13-fold improved activity towards SAM in comparison to the wild type. Molecular dynamic simulation indicates that the loops more flexible and more conducive to SAM release.

Automated summary

S-adenosyl-L-methionine (SAM) is an active form of methionine that plays a crucial role in various metabolic reactions. Methionine adenosyltransferase (MAT) is the enzyme responsible for the production of SAM from methionine and ATP. However, the lack of an efficient method for high-throughput detection of SAM has hindered directed evolution studies of MAT. In this study, a colorimetric method for directed evolution of MAT was established, leading to the discovery of variant I303V/Q22R with significantly improved activity towards SAM. Molecular dynamic simulation revealed that these variants had more flexible loops, facilitating the release of SAM.