Yoshida Ayako, Tomita Takeo, Kurihara Takeshi, Fushinobu Shinya, Kuzuyama Tomohisa, Nishiyama Makoto
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
J Mol Biol. 2007 Apr 27;368(2):521-36. doi: 10.1016/j.jmb.2007.02.017. Epub 2007 Feb 20.
Aspartate kinase (AK) catalyzes the first step of the biosynthesis of the aspartic acid family amino acids, and is regulated via feedback inhibition by end-products including Thr and Lys. To elucidate the mechanism of this inhibition, we determined the crystal structure of the regulatory subunit of AK from Corynebacterium glutamicum at 1.58 A resolution in the Thr-binding form, the first crystal structure of the regulatory subunit of alpha(2)beta(2)-type AK. The regulatory subunit contains two ACT domain motifs per monomer and is arranged as a dimer. Two non-equivalent ACT domains from different chains form an effector-binding unit that binds a single Thr molecule, and the resulting two effector-binding units of the dimer associate perpendicularly in a face-to-face manner. The regulatory subunit is a monomer in the absence of Thr but becomes a dimer by adding Thr. The dimerization is eliminated in mutant AKs with changes in the Thr-binding region, suggesting that the dimerization induced by Thr binding is a key step in the inhibitory mechanism of AK from C. glutamicum. A putative Lys-binding site and the inhibitory mechanism of CgAK are discussed.
天冬氨酸激酶（AK）催化天冬氨酸家族氨基酸生物合成的第一步，并受到包括苏氨酸（Thr）和赖氨酸（Lys）在内的终产物的反馈抑制调节。为阐明这种抑制机制，我们以1.58埃的分辨率确定了谷氨酸棒杆菌中天冬氨酸激酶调节亚基处于与苏氨酸结合形式时的晶体结构，这是α(2)β(2)型天冬氨酸激酶调节亚基的首个晶体结构。调节亚基每个单体包含两个ACT结构域基序，且以二聚体形式排列。来自不同链的两个不等价ACT结构域形成一个结合单个苏氨酸分子的效应物结合单元，二聚体产生的两个效应物结合单元以面对面的方式垂直缔合。调节亚基在没有苏氨酸时是单体，但通过添加苏氨酸变成二聚体。在苏氨酸结合区域发生变化的突变型天冬氨酸激酶中，二聚化被消除，这表明苏氨酸结合诱导的二聚化是谷氨酸棒杆菌中天冬氨酸激酶抑制机制的关键步骤。文中还讨论了一个假定的赖氨酸结合位点以及谷氨酸棒杆菌天冬氨酸激酶（CgAK）的抑制机制。
