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.

Heterologous expression and characterization of a thermoalkaliphilic SAM-synthetase from giant leucaena (Leucaena leucocephala subsp glabrata).

The cDNA encoding S-adenosylmethionine (SAM) synthetase was isolated from giant leucaena (Leucaena leucocephala subsp. glabrata) root tissue mRNA. Transcriptome data and 5'-RLM-RACE were used to obtain the transcript sequence and clone into the T7-expression vector pEt14b. N-terminal Histidine-tagged recombinant protein was expressed highly in Escherichia coli, purified and characterized by activity assays. A straightforward method using isocratic reverse-phase HPLC analysis (mobile phase: 0.02M o-phosphoric acid) of enzyme assays determined optimal enzyme activity at pH 10.0, 55 °C and 200 mM KCl. In addition to thermophilic activity, giant leucaena SAM-synthetase remains highly active in solutions containing up to 4 M KCl and accepts Na+ to some extent as a substitute for K+, a known required cofactor for SAM-synthetases. The enzyme followed Michaelis-Menten kinetics (Km = 1.82 mM, Kcat = 1.17 s-1, Vmax 243.9 µM. min-1) and was not inhibited by spermidine, spermine or nicotianamine. Giant leucaena SAM-synthetase is a highly tolerant enzyme to extreme conditions, suggesting further studies on plant SAM-synthetases.