Structure of Subtilosin A, an Antimicrobial Peptide from Bacillus subtilis with Unusual Posttranslational Modifications Linking Cysteine Sulfurs to r-Carbons of Phenylalanine and Threonine Karen Kawulka, ² Tara Sprules, ² Ryan T. McKay, ‡ Pascal Mercier, § Christopher M. Diaper, ² Peter Zuber, | and John C. Vederas* ,²,# Department of Chemistry, Department of Biochemistry, and National High Field NMR Centre (NANUC), UniVersity of Alberta, Edmonton, Alberta T6G 2G2, Canada, and EnVironmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health & Science UniVersity, BeaVerton, Oregon 97006 Received December 9, 2002; E-mail: john.vederas@ualberta.ca Bacteriocins are potent antimicrobial peptides produced by bacteria which are usually active against a limited spectrum of related organisms. Such ribosomally synthesized peptides are generally cationic, typically have from 25 to 60 amino acids, and fall into one of two major classes: either unmodified peptides (except for possible disulfide bridges) or lantibiotics. 1,2 The latter, for example, nisin A which is used in over 80 countries as a food preservative, are extensively posttranslationally modified. 2 Their biosynthesis involves enzymatic dehydration of serine or threonine residues to give dehydroalanine or dehydrobutyrine moieties, some of which then undergo Michael attack at the -position by nearby cysteine thiols to form monosulfide lanthionine bridges. 2,3 Our earlier work 4 on genes required for production of subtilosin A (1), a bacteriocin from Bacillus subtilis, indicated that the initially proposed structure 5 of this highly modified peptide required revision (Figure 1). Subsequent studies by Marx et al. suggested that thioether bridges were present: Cys13 to Phe22, Cys7 to Thr28, and Cys4 to Phe31. 6 However, the exact connectivity of these links remained uncertain, although bonds between sulfur and the aromatic rings of Phe22 and Phe31, as well as between sulfur and the -carbon of Thr28, were proposed. 6 We now report the determi- nation of the complete primary and three-dimensional solution structure of 1 using isotopic labeling and multidimensional NMR studies. The results demonstrate that in addition to having a cyclized peptide backbone, three cross-links are formed between sulfurs of cysteine and the R-positions of the two phenylalanines and the threonine. To the best of our knowledge, such posttranslational linkage of thiol to an R-carbon of an amino acid residue has not been previously observed in natural peptides or proteins. Thus, subtilosin A (1) belongs to a new class of bacteriocins. Although 1 can be readily produced and isolated in significant quantities (5-10 mg/L) from B. subtilis, it is highly resistant to proteinases and defies complete sequence analysis by Edman degradation or mass spectral examination. 4,5 To allow complete structural assignment by NMR, universally [ 13 C, 15 N]-enriched subtilosin A was prepared by fermentation of B. subtilis on a labeled peptone media generated from blue green algae (Anabaena sp.) grown on sodium [ 13 C]bicarbonate and sodium [ 15 N]nitrate as the sole carbon and nitrogen sources. 7 Levels of isotopic labeling of the peptone samples were determined to be 85-98% 13 C and 77-95% 15 N by combustion of small samples followed by atomic emission determination of 12 C/ 13 C and 14 N/ 15 N ratios. 8 Electrospray mass spectral analysis of the labeled subtilosin A derived from this peptone indicated comparable levels of labeling. NMR studies of labeled subtilosin A in methanol employed well- established, two- and three-dimensional techniques for protein analysis. 9 Complete assignment of all nitrogens, carbons, and hydrogens indicated that Phe22, Thr28, and Phe31 were modified at their R-carbons, which were fully substituted. To confirm this, universally labeled [ 13 C, 15 N]-L-phenylalanine and [ 13 C, 15 N]-L- threonine were added to separate fermentations of B. subtilis with standard unlabeled media. Examination of the resulting samples of 1 by 13 C-COSY shows the complete carbon connectivity pattern ² Department of Chemistry, University of Alberta. ‡ NANUC, University of Alberta. § Department of Biochemistry, University of Alberta. | Oregon Graduate Institute of Science and Technology. # Canada Research Chair in Bioorganic and Medicinal Chemistry. Figure 1. Linkages in subtilosin A (1), a cyclic peptide. Figure 2. NMR spectra of 1 labeled by U-[ 13 C, 15 N]Phe. Contour maps of (A) 13 C-COSY at 125 MHz showing Phe C (y-axis) to aromatic carbon (x-axis) correlations; (B) 13 C-COSY of Phe C (y-axis) to CR (x-axis) correlations; (C) 1 H, 13 C-plane from HNCA displaying intraresidue Phe CR (y-axis) to 1 H(x-axis), 15 N correlations. (D) 1 H, 13 C-HSQC demonstrating that only the Phe C (y-axis) have protons (x-axis) directly attached. The C and CR resonances of Phe22 and Phe31 are nearly overlapped (indistinguishable in panels A and B), but the two CR to NH correlations are clearly separated in panel C. Published on Web 03/29/2003 4726 9 J. AM. CHEM. SOC. 2003, 125, 4726-4727 10.1021/ja029654t CCC: $25.00 © 2003 American Chemical Society