Directed evolution of a bacterial sialidase and characterization of mechanism based inactivation of glycosidases
Bibliographic record
Abstract
Sialic acids are often found at the terminal positions on the glycan chains that adorn all vertebrate cells and glycoproteins.This prominent position confers an essential role to sialic acid residues in biology, evolution and disease propagation.The most widespread sialic acid family members are N-acetylneuraminic acid, N-glycolylneuraminic acid and Kdn, which is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid.Enzymes that catalyze the removal of carbohydrate linkages from biological molecules are called glycoside hydrolases (GHs).These enzymes have been categorized into more than 130 different families.Glycoside hydrolase family 33 (GH33) contains exo-sialidases (E.C. 3.2.1.18,neuraminidases), from both eukaryotes and prokaryotes, which catalyze the hydrolysis of sialic acid from glycoconjugates.Interestingly, subtle differences exist in both the structure of the particular sialic acid and its position of attachment to glycoconjugate chains between humans and other mammals.These differences are indicators of the unique aspects of human evolution, and are relevant to understanding an array of human conditions.The present thesis reports on routes that we explored to further unravel the importance of sialic acids.We developed tools to probe for various sialic acid structures such as Kdn.To this end, we constructed a random mutant library of the neuraminidase from the soil bacterium Micromonospora viridifaciens (MvNA) and identified a number of recurring mutations in the sialidase gene which lead to a more efficient hydrolysis of synthetic natural substrate analogues such as 8FMU α-Kdn-(2→6)β-D-Galp.We also using the available structure of wild type MvNA bound to the natural inhibitor, DANA, to identify amino acids potentially involved in recognition and binding to acetylated sialic acids and generated genetic libraries which we used along with positive and negative evolutionary screens to identify several clones capable of hydrolyzing Kdn glycosides more efficiently than Neu5Ac substrates.Kinetic studies on these clones allowed for determination of enzyme efficiencies and specificities.We also report our study of covalent inhibition of α-glucosidase from Saccharomyces cerevisiae (GH13).The measured pH-rate profiles for inhibition and reactivation as well as the corresponding catalytic and inhibitory proficiencies suggested that inhibition results from the formation of carbenium ions in the active site that are trapped rapidly by an enzymatic residue.
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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) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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".