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  • Title: Heterologous expression and characterization of a thermoalkaliphilic SAM-synthetase from giant leucaena (Leucaena leucocephala subsp glabrata).
    Author: Carrillo JT, Borthakur D.
    Journal: Plant Physiol Biochem; 2022 Jun 15; 181():42-49. PubMed ID: 35429803.
    Abstract:
    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.
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