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
Jeltsch, A.; Sobotta, T.; Pingoud, A.
Protein Engineering 9(5): 413-423
1996
ISSN/ISBN: 0269-2139 PMID: 8795041 Document Number: 464367
The EcoRV DNA methyltransferase (M cntdot EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M cntdot EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M cntdot 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 cntdot 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 cntdot EcoRV (Val20Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M cntdot 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 cntdot EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M cntdot EcoRV confirming the importance of these regions.