Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium <i>Aquifex aeolicus</i>
Bibliographic record
Abstract
A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (Delta19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95 degrees C or higher. Delta19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95 degrees C versus 2 h for PD, and values of kcat and Km for prephenate, which are twice those determined for PD at 80 degrees C. Low concentrations of guanidine-HCl activate enzyme activity, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation that proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady-state fluorescence intensity and its quenching by acrylamide in the presence of Gdn-HCl suggest that, of the two tryptophan residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried tryptophan is at the active site. Tyrosine is a feedback inhibitor of PD activity over a wide temperature range and enhances the cooperativity between subunits in the binding of prephenate. Properties of this thermostable PD are compared and contrasted with those of E. coli chorismate mutase-prephenate dehydrogenase and other mesophilic homologs.
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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) | 0.000 | 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".