Structural and functional characterization of Streptomyces plicatus B-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagensis
Bibliographic record
Abstract
A gene from Streptomyces plicatus encoding a $\\beta$-N-acetylhexosaminidase (SpHex) was sequenced and the encoded protein identified as a member of family 20 glycosyl hydrolases. This family includes human $\\beta$-N-acetylhexosaminidases whose deficiency results in various forms of $\\rm G\\sb{M2}$ gangliosidosis. Based upon the X-ray structure of Serratia marcescens chitobiase (SmChb), a three-dimensional model of SpHex was generated by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling derived structures of human $\\beta$-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb X-ray structure, putative SpHex active site residues were selected and mutated. An Arg162His mutation increased $K\\sb{m}$ 40 fold and reduced $V\\sb{max}$ 5 fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human $\\beta$-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate binding residue in a family 20 enzyme, a finding consistent with our 3-dimensional model of SpHex. Glu314Gln reduced $V\\sb{max}$ 296 fold, $K\\sb{m}$ 7 fold and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246Asn reduced $V\\sb{max}$ 2 fold and increased $K\\sb{m}$ only 1.2 fold, suggesting Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the X-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.
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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 | 0.000 |
| 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) | 0.001 | 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".