G. Vilkaitis et al., Functional roles of the conserved threonine 250 in the target recognition domain of HhaI DNA methyltransferase, J BIOL CHEM, 275(49), 2000, pp. 38722-38730
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.