Expression, Purification, and Characterization of a Galactofuranosyltransferase Involved in <i>Mycobacterium t</i> <i>uberculosis</i> Arabinogalactan Biosynthesis
Why this work is in the frame
A frame that forgets how it found something cannot be audited. These are the routes that admitted this work.
Bibliographic record
Abstract
The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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) | 0.001 | 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 it