Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the K m for substrate S- adenosyl- l- methionine (AdoMet), but a decrease in the K m for substrate DNA. This is reflected in minor changes in k cat/ K m for DNA, but in 10- to 100-fold reductions in k cat/ K m for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.