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†,‡
posted on 2004-03-30, 00:00authored byKaren E. Kawulka, Tara Sprules, Christopher M. Diaper, Randy M. Whittal, Ryan T. McKay, Pascal Mercier, Peter Zuber, John C. Vederas
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 [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 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 α-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 (α-R) and d
stereochemistry at Thr28 (α-S) and Phe31 (α-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 α-carbon were synthesized by reaction of the corresponding
α-alkoxy compounds with benzyl thiol and SnCl4. 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 α-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
α-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.