Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT
Bibliographic record
Abstract
Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase [also known as fatty acid:CoA ligase (AMP-forming); EC 6.2.1.3], as a 42–50kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43kDa C-terminal fragment and a 19kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62kDa form of the enzyme is present in vivo, but we utilized the proteolytic sensitivity of FadD to investigate its structure. Photoaffinity labelling experiments revealed that both intact FadD and the 43kDa fragment bound a long-chain fatty acid. Intact and cleaved FadD were also purified to determine the effect of cleavage on function. When using oleate as a substrate, cleaved FadD displayed 2-fold higher Km and Vmax values compared with intact FadD, but the catalytic efficiencies (kcat/Km) of the two forms were similar. This indicated that cleavage did not adversely affect enzyme activity. Proteolysis of FadD by OmpT was altered by the presence of oleate or ATP, both of which are ligands for the fatty acyl-CoA synthetase. This suggested that FadD undergoes ligand-induced conformational changes and implies that the region surrounding the cleavage site is mobile, a common characteristic of linker domains.
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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) | 0.001 | 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".