Mechanism of Aminoglycoside Antibiotic Kinase APH(3‘)-IIIa: Role of the Nucleotide Positioning Loop
Bibliographic record
Abstract
The aminoglycoside antibiotic resistance kinases (APHs) and the Ser/Thr/Tyr protein kinases share structural and functional homology but very little primary sequence conservation (<5%). A region of structural, but not amino acid sequence, homology is the nucleotide positioning loop (NPL) that closes down on the enzyme active site upon binding of ATP. This loop region has been implicated in facilitating phosphoryl transfer in protein kinases; however, there is no primary sequence conservation between APHs and protein kinases in the NPL. There is an invariant Ser residue in all APH NPL regions, however. This residue in APH(3')-IIIa (Ser27), an enzyme widespread in aminoglycoside-resistant Enterococci, Streptococci, and Staphylococci, directly interacts with the beta-phosphate of ATP through the Ser hydroxymethyl group and the amide hydrogen in the 3D structure of the enzyme. Mutagenesis of this residue to Ala and Pro supported a role for the Ser amide hydrogen in nucleotide capture and phosphoryl transfer. A molecular model of the proposed dissociative transition state, which is consistent with all of the available mechanistic data, suggested a role for the amide of the adjacent Met26 in phosphoryl transfer. Mutagenesis studies confirmed the importance of the amide hydrogen and suggest a mechanism where Ser27 anchors the ATP beta-phosphate facilitating bond breakage with the gamma-phosphate during formation of the metaphosphate-like transition, which is stabilized by interaction with the amide hydrogen of Met26. The APH NPL therefore acts as a lever, promoting phosphoryl transfer to the aminoglycoside substrate, with the biological outcome of clinically relevant antibiotic resistance.
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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".