Kamarthapu Venu, Rao Khareedu Venkateswara, Srinivas P N B S, Reddy G Bhanuprakash, Reddy Vudem Dashavantha
Center for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India.
Biochim Biophys Acta. 2008 Dec;1784(12):1949-58. doi: 10.1016/j.bbapap.2008.06.006. Epub 2008 Jun 19.
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.
S-腺苷甲硫氨酸（SAM）合成酶（EC 2.5.1.6）以L-甲硫氨酸和ATP作为底物催化S-腺苷甲硫氨酸的合成。首次从枯草芽孢杆菌中克隆出SAM合成酶基因（metE），并在异源宿主大肠杆菌中作为一种活性酶进行过量表达。尺寸排阻色谱（SEC）显示其分子量约为180 kDa，表明该酶是一种通过非共价相互作用稳定的同四聚体。SAM合成酶在pH 8.0和45℃时表现出最佳活性，需要二价阳离子Mg(2+)，并受到一价阳离子K(+)的刺激。该酶遵循顺序机制，V(max)为0.362微摩尔/分钟/毫克，对ATP和L-甲硫氨酸的K(m)分别为920微摩尔和260微摩尔。重组酶的尿素诱导的去折叠平衡显示出一个多态过程，包括部分去折叠的四聚体、结构二聚体、结构单体和完全去折叠的单体，这通过内源和外源荧光、圆二色性（CD）和SEC得到了证实。SEC中不存在三聚体表明该酶是二聚体的二聚体。SEC结果与尿素存在下的酶活性之间的一致性充分证明，只有具有亚基间活性位点的四聚体才表现出酶活性。
