Structure of Subtilosin A, a Cyclic Antimicrobial Peptide from Bacillus subtilis with Unusual Sulfur to α-Carbon Cross-Links:  Formation and Reduction of α-Thio-α-Amino Acid Derivatives^,

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.

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

“…There has been only one other report of sulfur to α-carbon linkages in a naturally occurring peptide, the antimicrobial subtilosin A (29). Subtilosin A is a 35-residue single-component bacteriocin that is backbone cyclized between its N and C termini.…”

Cysteine S -glycosylation, a new post-translational modification found in glycopeptide bacteriocins

“…The sulfur to a-carbon bridges of subtilosin A [26] and thuricin CD [27] are further testament to the versatility of cysteine thiols as substrates for post-translational modification enzymes. Peptide S-glycosylation adds an extra dimension to cysteine post-translational modifications, and natural glycodiversification may prove to be an evolutionarily facile process for generating new and improved bacteriocins.…”

Accessing chemical diversity from the uncultivated symbionts of small marine animals

“…55,56 In our hands, the conditions that worked best were as follows: peptide (150 µg, relative to amount estimated by weight) was suspended in 7:1 MeOH:H 2 O (500 µl) with NiCl 2 (1–2 mg) pre-dissolved in a 1.5 ml screw-cap reaction vial with stir bar. About a five-fold excess of dry NaBH 4 was added directly to the vial, which was immediately capped and place in an oil bath at 50°C for 30 min.…”