Binding of ATP as Well as Tetrahydrofolate Induces Conformational Changes in <i>Lactobacillus casei</i> Folylpolyglutamate Synthetase in Solution
Bibliographic record
Abstract
Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate to folate derivatives to form folate polyglutamates. FPGS is essential for folate biosynthesis in bacteria and retention of folate pools in eukaryotes. X-ray crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS suggest that binding of folate triggers a conformational change that activates FPGS. We used EPR and CD spectroscopy to further characterize the conformational change in the FPGS reaction. For EPR spectroscopy, two cysteine residues were introduced into FPGS by site-directed mutagenesis, K172C in the N-terminal domain and D345C in the C-terminal domain. The mutant protein was expressed, purified, and labeled with methanethiosulfonate. Addition of ATP, tetrahydrofolate, or 5,10-methylenetetrahydrofolate but not glutamate to FPGS showed broadening of EPR spectra, which is due to stronger spin-spin interactions, suggesting that both ATP and tetrahydrofolates cause a conformational change. ATP binding had an EPR spectrum distinct from that of tetrahydrofolate binding, indicating that it caused a different conformational change. When both ATP and THF were bound, the spectrum was identical to that seen when THF alone bound to the enzyme, showing that the THF-induced conformation was dominant. The spectral broadening suggests that the conformation change involves the two domains moving closer together, which is consistent with the rigid-body rotation of the C-terminal domain observed in the FPGS crystal structure with AMPPCP and 5,10-methylenetetrahydrofolate bound. No changes in the CD spectra were observed with the addition of FPGS substrates, suggesting that the conformational changes did not affect the secondary structure elements of the enzyme. These studies confirm the conformational change seen in the crystal structure by an independent method but also show that ATP binds to the free enzyme and affects its conformation.
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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 | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 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".