Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli

Abstract

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine using l-methionine and ATP as substrates. SAM synthetase gene ( metE) from Bacillus subtilis was cloned and over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~ 180 kDa, suggesting that the enzyme is a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity at pH 8.0 and 45 °C with the requirement of divalent cation Mg 2+, and stimulated by the monovalent cation K +. The enzyme followed sequential mechanism with a V max of 0.362 µmol/min/mg, and a K m of 920 µM and 260 µM for ATP and l-methionine, respectively. The urea-induced unfolding equilibrium of the recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer, structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsic and extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates that the enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in the presence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzyme activity.