Schluckebier G, Kozak M, Bleimling N, Weinhold E, Saenger W
Institut für Kristallographie Freie Universität Berlin, Germany.
J Mol Biol. 1997 Jan 10;265(1):56-67. doi: 10.1006/jmbi.1996.0711.
The crystal structures of the binary complexes of the DNA methyltransferase M.TaqI with the inhibitor Sinefungin and the reaction product S-adenosyl-L-homocysteine were determined, both at 2.6 A resolution. Structural comparison of these binary complexes with the complex formed by M.TaqI and the cofactor S-adenosyl-L-methionine suggests that the key element for molecular recognition of these ligands is the binding of their adenosine part in a pocket, and discrimination between cofactor, reaction product and inhibitor is mediated by different conformations of these molecules; the methionine part of S-adenosyl-L-methionine is located in the binding cleft, whereas the amino acid moieties of Sinefungin and S-adenosyl-L-homocysteine are in a different orientation and interact with the active site amino acid residues 105NPPY108. Dissociation constants for the complexes of M.TaqI with the three ligands were determined spectrofluorometrically. Sinefungin binds more strongly than S-adenosyl-L-homocysteine or S-adenosyl-L-methionine, with KD=0.34 microM, 2.4 microM and 2.0 microM, respectively.
测定了DNA甲基转移酶M.TaqI与抑制剂杀稻瘟菌素和反应产物S-腺苷-L-高半胱氨酸的二元复合物的晶体结构，分辨率均为2.6埃。将这些二元复合物与M.TaqI和辅因子S-腺苷-L-甲硫氨酸形成的复合物进行结构比较，结果表明，这些配体分子识别的关键要素是其腺苷部分在一个口袋中的结合，而辅因子、反应产物和抑制剂之间的区分是由这些分子的不同构象介导的；S-腺苷-L-甲硫氨酸的甲硫氨酸部分位于结合裂隙中，而杀稻瘟菌素和S-腺苷-L-高半胱氨酸的氨基酸部分则处于不同的方向，并与活性位点氨基酸残基105NPPY108相互作用。通过荧光光谱法测定了M.TaqI与这三种配体复合物的解离常数。杀稻瘟菌素的结合比S-腺苷-L-高半胱氨酸或S-腺苷-L-甲硫氨酸更强，其KD分别为0.34微摩尔、2.4微摩尔和2.0微摩尔。
