High-Throughput Screening and Directed Evolution of Methionine Adenosyltransferase from Escherichia coli.
Appl Biochem Biotechnol
; 195(7): 4053-4066, 2023 Jul.
Article
em En
| MEDLINE
| ID: mdl-36652094
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Escherichia coli
/
Metionina Adenosiltransferase
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article