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Mig.MthI from Methanobacterium thermoautotrophicum and MutY of Escherichia coli are both DNA mismatch glycosylases of the 'helix-hairpin-helix' (HhH) superfamily of DNA repair glycosylases; the former excises thymine from T/G, the latter adenine from A/G mismatches. The structure of MutY, in complex with its low molecular weight product, adenine, has previously been determined by X-ray crystallography. Surprisingly, the set of amino acid residues of MutY that are crucial for adenine recognition is largely conserved in Mig.MthI. Here we show that replacing two amino acid residues in the (modeled) thymine binding site of Mig.MthI (Leu187 to Gln and Ala50 to Val) changes substrate discrimination between T/G and A/G by a factor of 117 in favor of the latter (from 56-fold slower to 2.1-fold faster). The Ala to Val exchange also affects T/G versus U/G selectivity. The data allow a plausible model of thymine binding and of catalytic mechanism of Mig.MthI to be constructed, the key feature of which is a bidentate hydrogen bridge of a protonated glutamate end group (number 42) with thymine centers NH-3 and O-4, with proton transfer to the exocyclic oxygen atom neutralizing the negative charge that builds up in the pyrimidine ring system as the glycosidic bond is broken in a heterolytic fashion. The results also offer an explanation for why so many different substrate specificities are realized within the HhH superfamily of DNA repair glycosylases, and they widen the scope of these enzymes as practical tools.

Two amino acid replacements change the substrate preference of DNA mismatch glycosylase Mig.MthI from T/G to A/G