1pxq
From Proteopedia
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Structure of Subtilosin A
Overview
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.
About this Structure
1PXQ is a Single protein structure of sequence from Bacillus subtilis. Full crystallographic information is available from OCA.
Reference
Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives., Kawulka KE, Sprules T, Diaper CM, Whittal RM, McKay RT, Mercier P, Zuber P, Vederas JC, Biochemistry. 2004 Mar 30;43(12):3385-95. PMID:15035610
Page seeded by OCA on Wed Nov 21 00:12:43 2007
