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 methyltran sferase. 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 acti ve 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 aligne d to the common topology of the structures of the catalytic domains of M . HhaI and M . TaqI. In a complementary approach we have isolated n ine catalytically inactive single mutants. Five of these mutants conta in an amino acid exchange within the catalytic domain of M . EcoRV (Va l20-Ala, Lys81Arg, Cys192Arg, Asp193Gly, TrpZ31Arg). The Trp231Arg mut ant binds DNA similarly to wild-type M . EcoRV, but is catalytically i nactive. Hence this mutant behaves like a bona fide active site mutant , According to the structure prediction, Trp231 is located in a loop a t the putative active site of M . EcoRV. The other inactive mutants we re insoluble. They contain amino acid exchanges within the conserved a mino acid moths X, III or IV in M . EcoRV confirming the importance of these regions.