Dynamics and Reactivity in Thermus aquaticus N6-Adenine Methyltransferase

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• 作者：Juan Aranda ; Kirill Zinovjev ; Maite Roca ; I帽aki Tu帽贸n
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：November 19, 2014
• 年：2014
• 卷：136
• 期：46
• 页码：16227-16239
• 全文大小：740K
• ISSN：1520-5126

文摘

M.TaqI is a DNA methyltransferase from Thermus aquaticus that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the N6 position of an adenine, a process described only in prokaryotes. We have used full atomistic classical molecular dynamics simulations to explore the protein鈥揝AM鈥揇NA ternary complex where the target adenine is flipped out into the active site. Key protein鈥揇NA interactions established by the target adenine in the active site are described in detail. The relaxed structure was used for a combined quantum mechanics/molecular mechanics exploration of the reaction mechanism using the string method. According to our free energy calculations the reaction takes place through a stepwise mechanism where the methyl transfer precedes the abstraction of the proton from the exocyclic amino group. The methyl transfer is the rate-determining step, and the obtained free energy barrier is in good agreement with the value derived from the experimental rate constant. Two possible candidates to extract the leftover proton have been explored: a water molecule found in the active site and Asn105, a residue activated by the hydrogen bonds formed through the amide hydrogens. The barrier for the proton abstraction is smaller when Asn105 acts as a base. The reaction mechanisms can be different in other N6-DNA-methyltransferases, as determined from the exploration of the reaction mechanism in the Asn105Asp M.TaqI mutant.