Site-directed mutagenesis of the cysteine residues in the Pichia stipitis xylose reductase
Bibliographic record
Abstract
Xylose reductase catalyzes the reduction of xylose to xylitol and is known to play a pivotal role in pentose metabolism in yeasts. We previously showed that a cystein residue may be involved in binding of the coenzyme NADPH to the Pichia stipitis xylose reductase through chemical modification studies. The question arose as to which of the three cysteine residues in this enzyme may be involved in coenzyme binding. We cloned the XYL1 gene encoding xylose reductase from P. stipitis into the phagemid pEMBL18(+) suitable for site-directed mutagenesis. Each of the three cysteine residues (Cys19, Cys27 and Cys130) was individually mutated to serine. All three Cys-->Ser variants remained functional, but with reduced catalytic activity. Sensitivity of the P. stipitis xylose reductase to thiol-specific reagents was attributed to both Cys27 and Cys130 residues as substitution of either residue with Ser resulted in a significant but incomplete loss of sensitivity to PCMBS. The apparent Km values of the Cys variants for NADPH, NADH and xylose did not differ from those of the wild-type enzyme isolated from yeast by more than 4-fold. Our results suggest that none of the Cys residues are directly involved in NADPH binding, although Cys130 may reside in or near the coenzyme binding region and might play a role in coenzyme specificity.
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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".