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 approximately 16 and 
190-fold respectively, thus confirming the importance of this motif. However, 
these kinetic changes cannot account for the mutant's observed 10(5)-fold 
decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure 
(2.79 A resolution) shows the target cytosine to be positioned approximately 30 
degrees into the major groove, which is consistent with a major groove pathway 
for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to 
approximately +171 degrees, from a range of -95 degrees to -120 degrees in B 
DNA, and -77 degrees 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 A 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 A) 
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.