Structure and Function of Exo-.BETA.-glucosaminidase from Amycolatopsis orientalis
Bibliographic record
Abstract
We cloned and sequenced the gene encoding exo-β-glucosaminidase (GlcNase) from Amycolatopsis orientalis, and found that the gene has an open reading frame of 1032 residues with a calculated molecular mass of 110,557. The GlcNase has been classified as a member of family GH-2. Sequence alignments identified a group of GlcNase-related protein sequences forming a distinct subclass of family GH-2. When mono-N-acetylated chitotetraose [(GlcN)3-GlcNAc] was hydrolyzed by the enzyme, the GlcN unit was produced from the nonreducing end together with the transglycosylation products. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The rate of hydrolysis of the disaccharide, GlcN-GlcNAc, was somewhat lower than that of (GlcN)2, suggesting that the N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. Based on the time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)4], we obtained the values of binding free energy changes of +7.0, -2.9, -1.8, -0.9, -1.0 and -0.5 kcal/mol corresponding, respectively, to subsites (-2) (-1) (+1) (+2) (+3) (+4). Synergism resulting from mixing the A. orientalis GlcNase with Streptomyces sp. N174 endochitosanase was also observed when chitosan polysaccharide was used as the substrate. To identify the catalytic residue, mutations were introduced into the putative catalytic residues resulting in five mutated enzymes (D469A, D469E, E541D, E541Q and S468N/D469E) which were successfully produced. The four single mutants were devoid of enzymatic activity, indicating that Asp469 and Glu541 are essential for catalysis as predicted from sequence alignment of enzymes belonging to GH-2 family.
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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".