A novel dCMP methylase by engineering thymidylate synthase.
X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus caseiTS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 A further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic 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 orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.
Agarwalla
S
S
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143-0448, USA.
LaPorte
S
S
Liu
L
L
Finer-Moore
J
J
Stroud
R M
RM
Santi
D V
DV
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003843:Deoxycytidine Monophosphate / Q000737:chemistry / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D008745:Methylation; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; D003843:Deoxycytidine Monophosphate; C007267:2'-deoxyuridylic acid; C075106:deoxycytidylate methylase; D013940:Thymidylate Synthase