Asparagine 229 mutants of thymidylate synthase catalyze the methylation of 3-methyl-2'-deoxyuridine 5'-monophosphate.
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 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, l., & Santi, D. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8604-8608] but is a major determinant in substrate specificity [Liu, l., & Santi, D. V. (1993) Biochemistry 32, 9263-9267]. 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have kcat values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of dUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be more active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis. In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C--3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP in the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric interference of the 3-methyl group with the side chain of Asn 229.
Costi
P M
PM
Department of Biochemistry and Biophysics, University of California, San Francisco, 94143-0448, USA.
Liu
L
L
Finer-Moore
J S
JS
Stroud
R M
RM
Santi
D V
DV
D000595:Amino Acid Sequence; D001216:Asparagine; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
C099991:3-methyl-2'-deoxyuridine 5'-monophosphate; D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; D001216:Asparagine; D013940:Thymidylate Synthase