Du Quan, Wang Zhen, Schramm Vern L
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461
Proc Natl Acad Sci U S A. 2016 Mar 15;113(11):2916-21. doi: 10.1073/pnas.1522491113. Epub 2016 Feb 29.
Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys(1226) to cytosine (Cyt) C6, methyl transfer from S-adenosyl-l-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys(1226) attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.
人类DNA甲基转移酶1（DNMT1）通过将CpG甲基化特征从亲代链复制到子代链来维持DNA的表观遗传状态，从而在细胞分裂过程中产生可遗传的甲基化模式。DNMT1的催化机制包括半胱氨酸（Cys）（1226）对胞嘧啶（Cyt）C6的亲核攻击、从S-腺苷-L-甲硫氨酸（SAM）向Cyt C5的甲基转移以及从C5夺取质子以在DNA中形成甲基化的CpG。在此，我们报告了人类DNMT1催化反应主要过渡态（TS）的亚埃几何和静电结构。利用实验动力学同位素效应指导量子力学计算以求解TS结构。在Cys（1226）攻击Cyt C6之后发生甲基转移，并且甲基转移步骤是DNMT1的化学限速步骤。比较了TS和基态的静电势图，为TS处蛋白质与反应物之间的相互作用提供了电子基础。对DNMT1的TS的理解表明，利用类似分析获得对表观遗传修饰复杂反应的亚埃几何洞察是可能的。
