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Semi-rationally engineered variants of S-adenosylmethionine synthetase from Escherichia coli with reduced product inhibition and improved catalytic activity
- Wang, Xiu, Jiang, Yiqi, Wu, Mianbin, Zhu, Li, Yang, Lirong, Lin, Jianping
- Enzyme and microbial technology 2019 v.129 pp. 109355
- Escherichia coli, S-adenosylmethionine, active sites, adenosine triphosphate, catalytic activity, enzyme activity, hydrogen bonding, ligands, methionine adenosyltransferase, mutagenesis
- S-adenosylmethionine synthetase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM) from ATP and L-methionine. SAM is the major methyl donor for more than 100 transmethylation reactions. It is also a common cosubstrate involved in transsulfuration and aminopropylation. However, product inhibition largely restrains the activity of MAT and limits the enzymatic synthesis of SAM. In this research, the product inhibition of MAT from Escherichia coli was reduced via semi-rational modification. A triple variant (Variant III, I303 V/I65 V/L186 V) showed a 42-fold increase in Ki,ATP and a 2.08-fold increase in specific activity when compared to wild-type MAT. Its Ki,ATP was 0.42 mM and specific acitivity was 3.78 ± 0.19 U/mg. Increased Ki,ATP means reduced product inhibition which enhances SAM accumulation. The SAM produced by Variant III could reach to 3.27 mM while SAM produced by wild-type MAT was 1.62 mM in the presence of 10 mM substrates. When the residue in 104th of Variant III was further optimized by site-saturated mutagenesis, the specific activity of Variant IV (I303 V/I65 V/L186 V/N104 K) reached to 6.02 ± 0.22 U/mg at 37 °C, though the SAM concentration decreased to 2.68 mM with 10 mM substrates. Analysis of protein 3D structure suggests that changes in hydrogen bonds or other ligand interactions around active site may account for the variety of product inhibition and enzyme activity. The Variant III and Variant IV with reduced inhibition and improved enzyme activity in the study would be more suitable candidates for SAM production in the future.