Functional roles of the conserved threonine 250 in the target recognition domain of HhaI DNA methyltransferase

DNA cytosine-5-methyltransferase HhaI recognizes the GCGC sequence and flip s the inner cytosine out of DNA helix and into the catalytic site for methy lation. The 5'-phosphate of the flipped out cytosine is in contact with the conserved Thr-250 from the target recognition domain. We have produced 12 mutants of Thr-250 and examined their methylation potential in vivo. Six ac tive mutants were subjected to detailed biochemical and structural studies. Mutants with similar or smaller side chains (Ser, Cys, and Gly) are very s imilar to wild-type enzyme in terms of steady-state kinetic parameters k(ca t), K-M(DNA), R-m(AdoMet). In contrast, the mutants with builder side chain s (Asn, Asp, and His) show increased K-m values for both substrates. Fluore scence titrations and stopped flow kinetic analysis of interactions with du plex oligonucleotides containing 2-aminopurine at the target base position indicate that the T250G mutation leads to a more polar but less solvent-acc essible position of the flipped out target base. The x-ray structure of the ternary M.HhaI(T250G).DNA.AdoHcy complex shows that the target cytosine is locked in the catalytic center of enzyme. The space created by the mutatio n is filled by water molecules and the adjacent DNA backbone atoms dislocat e slightly toward the missing side chain. In aggregate, our results suggest that the side chain of Thr-250 is involved in constraining the conformatio n the DNA backbone and the target base during its rotation into the catalyt ic site of enzyme.