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
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
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DOI：
10.1021/bi0359527
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发表时间：
2004-03-30
期刊：
影响因子：
2.9
通讯作者：
Vederas, JC
中科院分区：
文献类型：
--
作者：
Kawulka, KE;Sprules, T;Vederas, JC
枯萎病A（1）（来自枯草芽孢杆菌的细菌素）的完整原发性和三维溶液结构通过使用同位素标记的[C-13，N-15]介质从Anabaena SP衍生而成的多维NMR研究确定。在碳酸氢钠和[N-15]硝酸盐上生长。还通过使用其他未标记的介质的[U-C-13，N-15] -L-苯丙氨酸和[U-C-13，N-15] -L-硫代氨酸的单独掺入也会生成1个的其他样品。结果表明，除了具有环化肽主链（N和C末端之间的酰胺）外，在Cys13，Cys7和Cys4的硫硫和Phe22，Thr28和Phe3L的位置之间还形成了三个交联。 ， 分别。除三个修改后的三个残基的立体定义性通过与镍氢镍完全脱硫，酸水解为组成型氨基酸，并将其转化为相应的五氟丙酰胺异丙基酯，以实现三个修饰的立体化。通过对八回合ARIA结构计算的八个可能的立体异构体中的每一个中的每一个，从相同的NMR峰值文件和分配开始，确定了修改后的残基的立体定义。具有ph22（alpha-r）的L StereChernion的立体异构体，在Thr28（Alpha-S）（Alpha-S）和Phe31（Alpha-S）（alpha-s）（LDD ISOMER）符合NMR数据，符合NMR数据，为结构的最低能量家族提供了最佳的RMSD， 。因此，不寻常的Thio链接的生化形成是在PHE22处的构型净保留以及THR28和PHE3L的反转的。通过相应的α-烷氧化合物与苄基硫醇和SNCL4的反应合成了带有硫化物部分的模型氨基酸衍生物。与先前报道的定位化学对类固醇硫化物的类似反应相比，将其纯净的立体异构体和用镍硼化的脱硫化的分离表明，这种化合物的还原进行了呈现。然而，将枯草丝A的脱硫A与抗微生物剂无活性的环状肽14的脱硫化发生在Phe22和Thr28的Alpha-Carbons的反转时，并且在Phe31时保留4：1。这表明脱硫反应是通过N-酰胺亚胺进行的，而周围肽的结构控制了还原的几何形状。硫醇与氨基酸残基的A-碳的翻译后连接在核糖体合成的肽或蛋白质中是前所未有的，在次生代谢产物中非常罕见。枯草脂蛋白A（1）代表一类新的细菌素。
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 [C-13,N-15]medium derived from Anabaena sp. grown on sodium [C-13]bicarbonate and [N-15]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-C-13,N-15]-L-phenylalanine and [U-C-13,N-15]-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 a.-positions of Phe22, Thr28, and Phe3l, respectively. The stereochernistry of all residues in I 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 stereochernistry 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 stereochernistry at Phe22 (alpha-R) and D stereochermstry 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 Phe3l. 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 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 stereochernistry 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 a-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.