Mutation of asparagine 229 to aspartate in thymidylate synthase converts the enzyme to a deoxycytidylate methylase.
The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate dUMP. The Asn 229 to Asp mutant of Lactobacillus casei thymidylate synthase (TS N229D) has been prepared, purified, and investigated. Steady-state kinetic parameters of TS N229D show 3.5- and 10-fold increases in the Km values of CH2H4folate and dUMP, respectively, and a 1000-fold decrease in kcat. Most important, the Asp 229 mutation changes the substrate specificity of TS to an enzyme which recognizes and methylates dCMP in preference to dUMP. With TS N229D the Km for dCMP is bout 3-fold higher than for dUMP, and the Km for CH2H4folate is increased about 5-fold; however, the kcat for dCMP methylation is 120-fold higher than that for dUMP methylation. Specificity for dCMP versus dUMP, as measured by kcat/Km, changes from negligible with wild-type TS to about a 40-fold increase with TS N229D. TS N229D reacts with CH2H4folate and FdUMP or FdCMP to form ternary complexes which are analogous to the TS-FdUMP-CH2H4folate complex. From what is known of the mechanism and structure of TS, the dramatic change in substrate specificity of TS N229D is proposed to involve a hydrogen bond network between Asp 229 and the 3-N and 4-NH2 of the cytosine heterocycle, causing protonation of the 3-N and stabilization of a reactive imino tautomer. A similar mechanism is proposed for related enzymes which catalyze one-carbon transfers to cytosine heterocycles.
Liu
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Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*
D001224:Aspartic Acid; D001216:Asparagine; C075106:deoxycytidylate methylase; D013940:Thymidylate Synthase