Kinetic characterization of annotated glycolytic enzymes present in cellulose-fermenting Clostridium thermocellum suggests different metabolic roles
Bibliographic record
Abstract
Abstract Background The efficient production of sustainable biofuels is important for the reduction of greenhouse gas emissions. Clostridium thermocellum ATCC 27405 is a candidate for ethanol production from lignocellulosic biomass using consolidated bioprocessing. Fermentation of cellulosic biomass goes through an atypical glycolytic pathway in this thermophilic bacterium, with various glycolytic enzymes capable of utilizing different phosphate donors, including GTP and inorganic pyrophosphate (PP i ), in addition to or in place of the usual ATP. C . thermocellum contains three annotated phosphofructokinases (PFK) genes, the expression of which have all been detected through proteomics and transcriptomics. Pfp (Cthe_0347) was previously characterized as pyrophosphate dependent with fructose-6-phosphate (F6P) as its substrate. Results We now demonstrate that this enzyme can also phosphorylate sedoheptulose-7-phosphate (an intermediate in the pentose phosphate pathway), with the V max and K m of F6P being approximately 15 folds higher and 43 folds lower, respectively, in comparison to sedoheptulose-7-phosphate. Purified PfkA shows preference for GTP as the phosphate donor as opposed to ATP with a 12.5-fold difference in K m values while phosphorylating F6P. Allosteric regulation is a factor at play in PfkA activity, with F6P exhibiting positive cooperativity, and an apparent requirement for ammonium ions to attain maximal activity. Phosphoenolpyruvate and PP i were the only inhibitors for PfkA determined from the study, which corroborates what is known about enzymes from this subfamily. The activation or inhibition by these ligands lends support to the argument that glycolysis is regulated by metabolites such as PP i and NH 4 + in the organism. PfkB, showed no activity with F6P, but had significant activity with fructose, while utilizing either ATP or GTP, making it a fructokinase. Rounding out the upper glycolysis pathway, the identity of the fructose-1,6-bisphosphate aldolase in the genome was verified and reported to have substantial activity with fructose-1,6-bisphosphate, in the presence of the divalent ion, Zn 2+ . Conclusion These findings along with previous proteomic data suggest that Pfp, plays a role in both glycolysis and the pentose phosphate pathway, while PfkA and PfkB may phosphorylate sugars in glycolysis but is responsible for sugar metabolism elsewhere under conditions outside of growth on sufficient cellobiose.
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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 | 0.001 | 0.001 |
| 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".