Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadeno sine (OG,A) mismatches in DNA [Michaels et al, (1992) Proc. Natl. Acad. Sci , U.S.A. 89, 7022-7025]. MutY prevents DNA mutations resulting from the mis incorporation of A opposite OG by using N-glycosylase activity to remove th e adenine base, An interesting feature of MutY is that it contains a [4Fe-4 S](2+) cluster that has been shown to play an important role in substrate r ecognition [Porello, S. L., Cannon, M. J., David, S. S. (1998) Biochemistry 37, 6465-6475]. Herein, we have used site-directed mutagenesis to individu ally replace the cysteine ligands to the [4Fe-4S](2+) cluster of E. coli Mu tY with serine, histidine, and alanine. The extent to which the various mut ations reduce the levels of protein overexpression suggests that coordinati on of the [4Fe-4S](2+) cluster provides stability to MutY in vivo. The abil ity of the mutated enzymes to bind to a substrate analogue DNA duplex and t heir in vivo activity were evaluated, Remarkably, the effects; are both sub stitution and position dependent. For example, replacement of cysteine 199 with histidine provides a mutated enzyme that is expressed at high levels a nd exhibits DNA binding and in vivo activity similar to the WT enzyme. Thes e results suggest that histidine coordination to the iron-sulfur cluster ma y be accommodated at this position in MutY. In contrast, replacement of cys teine 192 with histidine results in less efficient DNA binding and in vivo activity compared to the WT enzyme without affecting levels of overexpressi on. The results from the site-directed mutagenesis suggest that the structu ral properties of the iron-sulfur cluster coordination domain are important for both substrate DNA recognition and the in vivo activity of MutY.