High-Throughput Screening and Directed Evolution of Methionine Adenosyltransferase from Escherichia coli.
Chenqi CaoKaili NieHaijun XuLuo LiuPublished in: Applied biochemistry and biotechnology (2023)
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.
Keyphrases
- escherichia coli
- high throughput
- amino acid
- wild type
- gold nanoparticles
- single cell
- hydrogen peroxide
- gene expression
- genome wide
- nitric oxide
- mass spectrometry
- staphylococcus aureus
- pseudomonas aeruginosa
- sensitive detection
- loop mediated isothermal amplification
- candida albicans
- biofilm formation
- multidrug resistant
- real time pcr