The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI

Fa Kuen Shieh, Ben Youngblood, Norbert O. Reich

研究成果: 雜誌貢獻期刊論文同行評審

42 引文 斯高帕斯(Scopus)

摘要

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased ∼16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 105-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 Å resolution) shows the target cytosine to be positioned ∼30° into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar χ angle is rotated to approximately +171°, from a range of -95° to -120° in B DNA, and -77° in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2′-endo-C3′-exo (South conformation), in contrast to the previously reported C3′-endo (North conformation) described for the original 2.70 Å resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 Å) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4′-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.

原文???core.languages.en_GB???
頁(從 - 到)516-527
頁數12
期刊Journal of Molecular Biology
362
發行號3
DOIs
出版狀態已出版 - 22 9月 2006

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