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

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 mutantʹs 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.