Structural and kinetic characterization of an acetoacetyl-Coenzyme A: acetate Coenzyme A transferase from the extreme thermophile <i>Thermosipho melanesiensis</i>
Bibliographic record
Abstract
Family 1 Coenzyme A transferases (CtfAB) from the extremely thermophilic bacterium, Thermosipho melanesiensis, has been used for in vivo acetone production up to 70°C. This enzyme has tentatively been identified as the rate-limiting step, due to its relatively low-binding affinity for acetate. However, existing kinetic and mechanistic studies on this enzyme are insufficient to evaluate this hypothesis. Here, kinetic analysis of purified recombinant T. melanesiensis CtfAB showed that it has a ping-pong bi-bi mechanism typical of Coenzyme A (CoA) transferases with Km values for acetate and acetoacetyl-CoA of 85 mM and 135 μM, respectively. Product inhibition by acetyl-CoA was competitive with respect to acetoacetyl-CoA and non-competitive with respect to acetate. Crystal structures of wild-type and mutant T. melanesiensis CtfAB were solved in the presence of acetate and in the presence or absence of acetyl-CoA. These structures led to a proposed structural basis for the competitive and non-competitive inhibition of acetyl-CoA: acetate binds independently of acetyl-CoA in an apparent low-affinity binding pocket in CtfA that is directly adjacent to a catalytic glutamate in CtfB. Similar to other CoA transferases, acetyl-CoA is bound in an apparent high-affinity binding site in CtfB with most interactions occurring between the phospho-ADP of CoA and CtfB residues far from the acetate binding pocket. This structural-based mechanism also explains the organic acid promiscuity of CtfAB. High-affinity interactions are predominantly between the conserved phospho-ADP of CoA, and the variable organic acid binding site is a low-affinity binding site with few specific interactions.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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 teacher head, 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".