Structure of Subtilosin A, a Cyclic Antimicrobial Peptide from Bacillus subtilis with Unusual Sulfur to -Carbon Cross-Links:
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- 作者：Karen E. Kawulka ; Tara Sprules ; Christopher M. Diaper ; Randy M. Whittal ; Ryan T. McKay ; Pascal Mercier ; Peter Zuber ; and John C. Vederas
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 30, 2004
- 年：2004
- 卷：43
- 期：12
- 页码：3385 - 3395
- 全文大小：262K
- 年卷期：v.43,no.12(March 30, 2004)
- ISSN：1520-4995
文摘
The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocinfrom Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced usingisotopically labeled [13C,15N]medium derived from Anabaena sp. grown on sodium [13C]bicarbonate and[15N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-13C,15N]-L-phenylalanine and [U-13C,15N]-L-threonine using otherwise unlabeled media. The results demonstrate thatin addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links areformed between the sulfurs of Cys13, Cys7, and Cys4 and the 
-positions of Phe22, Thr28, and Phe31,respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmedto 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 MSanalysis. The stereochemistry at the modified residues was determined by subjecting each of the eightpossible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMRpeak files and assignments. The stereoisomer with the L stereochemistry at Phe22 (-R) and Dstereochemistry at Thr28 (-S) and Phe31 (-S) (LDD isomer) fit the NMR data, giving the lowest energyfamily of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceedswith net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acidderivatives bearing a sulfide moiety at the -carbon were synthesized by reaction of the corresponding-alkoxy compounds with benzyl thiol and SnCl4. Separation of their pure stereoisomers and desulfurizationwith nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, incontrast 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 -carbons of Phe22 and Thr28 and with 4:1 retention atPhe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structureof the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, andvery rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.
-positions of Phe22, Thr28, and Phe31,respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmedto 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 MSanalysis. The stereochemistry at the modified residues was determined by subjecting each of the eightpossible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMRpeak files and assignments. The stereoisomer with the L stereochemistry at Phe22 (-R) and Dstereochemistry at Thr28 (-S) and Phe31 (-S) (LDD isomer) fit the NMR data, giving the lowest energyfamily of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceedswith net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acidderivatives bearing a sulfide moiety at the -carbon were synthesized by reaction of the corresponding-alkoxy compounds with benzyl thiol and SnCl4. Separation of their pure stereoisomers and desulfurizationwith nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, incontrast 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 -carbons of Phe22 and Thr28 and with 4:1 retention atPhe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structureof the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, andvery rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.