Kinetic and Structural Evaluation of Selected Active\nSite Mutants of the <i>Aspergillus fumigatus</i> KDNase\n(Sialidase)
Bibliographic record
Abstract
<i>Aspergillus fumigatus</i> is an airborne fungal pathogen.\nWe previously cloned and characterized an <i>exo</i>-sialidase\nfrom <i>A. fumigatus</i> and showed that it preferred 2-keto-3-deoxynononic\nacid (KDN) as a substrate to <i>N</i>-acetylneuraminic acid\n(Neu5Ac). The purpose of this study was to investigate the structure–function\nrelationships of critical catalytic site residues. Site-directed mutagenesis\nwas used to create three mutant recombinant enzymes: the catalytic\nnucleophile (Y358H), the general acid/base catalyst (D84A), and an\nenlargement of the binding pocket to attempt to accommodate the <i>N</i>-acetyl group of Neu5Ac (R171L). Crystal structures for\nall enzymes were determined. The D84A mutation had an effect in decreasing\nthe activity of AfKDNase that was stronger than that of the same mutation\nin the structurally similar sialidase from the bacterium <i>Micromonospora\nviridifaciens</i>. These data suggest that the catalytic acid\nis more important in the reaction of AfKDNase and that catalysis is\nless dependent on nucleophilic or electrostatic stabilization of the\ndeveloping positive charge at the transition state for hydrolysis.\nRemoval of the catalytic nucleophile (Y358H) significantly lowered\nthe activity of the enzyme, but this mutant remained a retaining glycosidase\nas demonstrated by nuclear magnetic resonance spectroscopic analysis.\nThis is a novel finding that has not been shown with other sialidases.\nKinetic activity measured at pH 5.2 revealed that R171L had higher\nactivity on a Neu5Ac-based substrate than wild-type KDNase; hence,\nleucine in place of arginine in the binding pocket improved catalysis\ntoward Neu5Ac substrates. Hence, whether a sialidase is primarily\na KDNase or a neuraminidase is due in part to the presence of an amino\nacid that creates a steric clash with the <i>N</i>-acetyl\ngroup.
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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 | 0.001 |
| 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) | 0.005 | 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".