Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase.
Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H(4)folate are products of the reaction. Production of H(4)folate rather than H(2)folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH(2)H(4)folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.
Griffin
Jonathon
J
National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba R3E 3R2, Canada.
Roshick
Christine
C
Iliffe-Lee
Emma
E
McClarty
Grant
G
D000595:Amino Acid Sequence; D002384:Catalysis / Q000187:drug effects; D002692:Chlamydia trachomatis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D015195:Drug Design; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005415:Flavins / Q000378:metabolism* / Q000494:pharmacology; D005492:Folic Acid / Q000378:metabolism; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D005415:Flavins; D011994:Recombinant Proteins; D013938:Thymidine Monophosphate; D009249:NADP; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase