Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli.
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 degrees 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 micromol/min/mg, and a K(m) of 920 microM and 260 microM 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.
Kamarthapu
Venu
V
Center for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India.
Rao
Khareedu Venkateswara
KV
Srinivas
P N B S
PN
Reddy
G Bhanuprakash
GB
Reddy
Vudem Dashavantha
VD
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000235:genetics; D011188:Potassium / Q000737:chemistry; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014508:Urea; D008716:Methionine Adenosyltransferase; D008274:Magnesium; D011188:Potassium