Wolters Stefan, Neeb Manuel, Berim Anna, Schulze Wischeler Johannes, Petersen Maike, Heine Andreas
Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
Acta Crystallogr D Biol Crystallogr. 2013 May;69(Pt 5):888-900. doi: 10.1107/S0907444913002874. Epub 2013 Apr 19.
Coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum (Linaceae) catalyzes the unusual methylation of the side-chain hydroxyl group of coniferyl alcohol. The protein was heterologously expressed in Escherichia coli as a hexahistidine derivative and purified for crystallization. Diffracting crystals were obtained of the pure protein and of its selenomethionine derivative, as well as of complexes with coniferyl alcohol and with S-adenosyl-L-homocysteine together with coniferyl alcohol 9-O-methyl ether (PDB entries 4ems, 4e70 and 4evi, respectively). The X-ray structures show that the phenylpropanoid binding mode differs from other phenylpropanoid O-methyltransferases such as caffeic acid O-methyltransferase. Moreover, the active site lacks the usually conserved and catalytic histidine residue and thus implies a different reaction mode for methylation. Site-directed mutagenesis was carried out to identify critical amino acids. The binding order of coniferyl alcohol and S-adenosyl-L-methionine was investigated by isothermal titration calorimetry experiments.
来自亚麻科植物小花亚麻（Linum nodiflorum）的松柏醇9-O-甲基转移酶催化松柏醇侧链羟基的异常甲基化反应。该蛋白以六组氨酸衍生物的形式在大肠杆菌中进行异源表达，并经纯化用于结晶。获得了该纯蛋白及其硒代甲硫氨酸衍生物的衍射晶体，以及与松柏醇、与S-腺苷-L-高半胱氨酸以及松柏醇9-O-甲基醚形成的复合物的衍射晶体（分别对应PDB编号4ems、4e70和4evi）。X射线晶体结构表明，该苯丙烷类化合物的结合模式不同于其他苯丙烷类O-甲基转移酶，如咖啡酸O-甲基转移酶。此外，活性位点缺少通常保守的催化组氨酸残基，因此意味着甲基化反应模式不同。通过定点诱变来鉴定关键氨基酸。通过等温滴定量热实验研究了松柏醇和S-腺苷-L-甲硫氨酸的结合顺序。
