Knappe Thomas A, Linne Uwe, Robbel Lars, Marahiel Mohamed A
Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
Chem Biol. 2009 Dec 24;16(12):1290-8. doi: 10.1016/j.chembiol.2009.11.009.
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 (Gly1, Arg11, Val12, and Ile13) 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.
Capistruin是一种由19个氨基酸组成的核糖体合成套索肽,由泰国伯克霍尔德菌中的capABCD基因簇编码。它由一个N端9个氨基酸的大环内酰胺环组成,10个氨基酸的C端尾巴穿过该环。我们利用大肠杆菌中的异源Capistruin生产系统,生成了前体蛋白CapA的48个突变体,以深入了解Capistruin的生物合成。发现套索序列中只有4个残基(Gly1、Arg11、Val12和Ile13)对成熟至关重要。Capistruin F16A/F18A的串联质谱裂解研究证明Arg15负责捕获C端尾巴。用丙氨酸取代Arg15和Phe16得到一种温度敏感的Capistruin衍生物,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总体特异性较低以及重要的结构-稳定性相关性。