Insights into the Biosynthesis and Stability of the Lasso Peptide Capistruin
Insights into the Biosynthesis and Stability of the Lasso Peptide Capistruin
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DOI：
10.1016/j.chembiol.2009.11.009
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发表时间：
2009-12-24
影响因子：
--
通讯作者：
Marahiel, Mohamed A.
中科院分区：
文献类型：
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作者：
Knappe, Thomas A.;Linne, Uwe;Marahiel, Mohamed A.
Capistruin 是一种由 19 个残基核糖体合成的套索肽，由泰国伯克霍尔德杆菌的 capABCD 基因簇编码。它由 N 端 9 残基大环内酰胺环组成，10 残基 C 端尾部穿过该环。利用大肠杆菌中的异源 Capistruin 生产系统，我们生成了 48 个前体蛋白 CapA 突变体，以深入了解 Capistruin 的生物合成。发现套索序列中只有 4 个残基（Glyl、Arg11、Vai12 和 lie13）对于成熟至关重要。 Capistruin F16A/F18A 的串联质谱裂解研究证明 Arg15 负责捕获 C 末端尾部。用丙氨酸取代 Arg15 和 Phe16 揭示了一种温度敏感的 Capistruin 衍生物，该衍生物在加热时会展开成支链环肽。总之，我们的全局诱变方法揭示了生物合成机制的整体特异性较低以及重要的结构稳定性相关性。
Capistruin is a 19-residue ribosomally synthesized lasso peptide encoded by the capABCD gene cluster in Burkholderia thailandensis. It is composed of an N-terminal 9-residue macrolactam ring, through which the 10-residue C-terminal tail is threaded. Using a heterologous capistruin production system in Escherichia coli, we have generated 48 mutants of the precursor protein CapA to gain insights into capistruin biosynthesis. Only 4 residues (Glyl, Arg11, Vai12, and lie13) of the lasso sequence were found to be critical for maturation. Tandem mass spectrometric fragmentation studies of capistruin F16A/F18A proved Arg15 to be responsible for the trapping of the C-terminal tail. Substituting Arg15 and Phe16 by alanine revealed a temperature-sensitive capistruin derivative, which unfolds into a branched cyclic peptide upon heating. In conclusion, our global mutagenic approach revealed a low overall specificity of the biosynthetic machinery and important structure-stability correlations.