Catalytic Mechanism of Chlamydia trachomatis Flavin-dependent Thymidylate Synthase
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".