A NOVEL DCMP METHYLASE BY ENGINEERING THYMIDYLATE SYNTHASE

X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that t here is a steric clash between the 4-NH2 of dCMP and His 199, a residu e which normally H-bonds to the 4-O of dUMP but is not essential for a ctivity. As a result, the cytosine moiety of dCMP is displaced from th e active site and the catalytic thiol is moved from the C6 of the subs trate about 0.5 Angstrom further than in the wild-type TS-dUMP complex . We reasoned that combining the N229D mutation with mutations at resi due 199 which did not impinge on the 4-NH2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prep ared several TS N229D mutants and characterized their steady state kin etic parameters. TS H199A/N229D showed a 10(11) change in specificity for methylation of dCMP versus dUMP. The structures of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position and orientat ion of bound dCMP closely approaches that of dUMP in wild-type TS, whe reas dUMP was displaced from the optimal catalytic binding site.