非核糖体肽Skyllamycin B生物合成中I型硫酯酶底物杂泛性的初步研究

[背景] Skyllamycins是一类从链霉菌中发现的具有血小板生长因子抑制和生物膜抑制作用的非核糖体肽类，其环肽环合反应是由非核糖体肽合成酶中的硫酯酶功能域催化完成。[目的] 克隆和表达Skyllamycin非核糖体肽合成酶最后一个模块中的硫酯酶（Skyxy-TE）基因，合成Skyxy-TE底物类似物，通过体外催化实验表征Skyxy-TE的底物杂泛性。[方法] 采用Ligation Independent Cloning（LIC）方法，从一株含有Skyllamycin B生物合成基因簇的链霉菌Streptomyces sp.PKU-MA01239中克隆和表达skyxy-TE，通过镍离子柱亲和层析纯化Skyxy-TE。运用固相多肽合成法合成2个底物类似物1和2，进行Skyxy-TE的体外催化实验。[结果] 通过对Skyxy-TE的表达纯化，获得了纯度较好的可溶性蛋白；通过固相多肽合成，得到了能够模拟Skyllamycin B底物类似物的化合物1和2，硫酯酶蛋白体外催化化合物1和2得到了化合物3和4，化合物3和4通过核磁共振和高分辨质谱确认为环肽。[结论] Skyllamycin B生物合成中Skyxy-TE表现出一定的底物杂泛性，可以识别底物类似物催化环化反应，该研究为将来利用化学-酶联法制备更多环肽类似物提供了依据。

Characterization of the substrate promiscuity of type I thioesterase in skyllamycin B biosynthesis

[Background] Skyllamycins are nonribosomal cyclic depsipeptides that possess inhibitory activity against the platelet-derived growth factor (PDGF) signaling pathway and antibiofilm activity. The cyclization reaction in skyllamycin biosynthesis is catalyzed by the thioesterase domain. [Objective] To characterize the substrate promiscuity of thioesterase domain, we cloned and expressed the thioesterase-encoding gene, synthesized substrate mimics and carried out the cyclization reactions in vitro. [Methods] The thioesterase domain (Skyxy-TE)-encoding gene was cloned from Streptomyces sp. PKU-MA01239 with ligation-independent cloning method, Skyxy-TE was purified by Ni-NTA affinity chromatography, and substrate mimics were synthesized by using solid-phase peptide synthesis (SPPS). [Results] Soluble Skyxy-TE was obtained and purified to homogeneity, and two substrate mimics 1 and 2 were synthesized by SPPS. Cyclization reactions were carried out in vitro, leading to the production of two cyclized peptides 3 and 4, whose structures were elucidated by NMR and HRESIMS analysis. [Conclusion] Skyxy-TE showed substrate promiscuity by catalyzing two substrate mimics to generate cyclization products, which facilitates the generation of more cyclized peptide analogues by chemoenzymatic synthesis in the future.