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

• Main Authors: Borthakur, D. (Author), Carrillo, J.T (Author)
• Format: Article
• Language: English
• Published: Elsevier Masson s.r.l. 2022
• Subjects: Heterologous expression; Methionine adenosyltransferase (MAT); Plant enzyme; Polyamine; Recombinant; S-adenosylmethionine (SAM); Thermophilic
• Online Access: View Fulltext in Publisher

 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. © 2022 The Authors