Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M . EcoRV) is an a-adenine methyltransferase. We have used two different programs to predict the secondary structure of M . EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis, 29 neutral mutations of M . EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M . HhaI and M . TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M . EcoRV (Val20-Ala, Lys81Arg, Cys192Arg, Asp193Gly, TrpZ31Arg). The Trp231Arg mutant binds DNA similarly to wild-type M . EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant, According to the structure prediction, Trp231 is located in a loop at the putative active site of M . EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid moths X, III or IV in M . EcoRV confirming the importance of these regions.