Structure of Subtilosin A, a Cyclic Antimicrobial Peptide from <i>Bacillus subtilis</i> with Unusual Sulfur to α-Carbon Cross-Links: Formation and Reduction of α-Thio-α-Amino Acid Derivatives<sup>,</sup>
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Bibliographic record
Abstract
The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]-L-phenylalanine and [U-(13)C,(15)N]-L-threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis. The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments. The stereoisomer with the l stereochemistry at Phe22 (alpha-R) and d stereochemistry at Thr28 (alpha-S) and Phe31 (alpha-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acid derivatives bearing a sulfide moiety at the alpha-carbon were synthesized by reaction of the corresponding alpha-alkoxy compounds with benzyl thiol and SnCl(4). Separation of their pure stereoisomers and desulfurization with nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, in contrast to the previously reported retention of stereochemistry for analogous reaction of steroidal sulfides. However, desulfurization of subtilosin A to cyclic peptide 14, which is inactive as an antimicrobial agent, occurs with inversion of stereochemistry at the alpha-carbons of Phe22 and Thr28 and with 4:1 retention at Phe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structure of the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.
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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 it