Non-ribosomal peptides (NRPs) are biosynthesized on non-ribosomal peptides synthetase (NRPS) complexes, of which a C-terminal releasing domain commonly offloads the products. Interestingly, a dedicated releasing domain is absent in surugamides (SGM) NRPS, which directs the biosynthesis of cyclic octapeptides, SGM-A to -E, and the linear decapeptide, SGM-F. Here, we confirmed that surE is essential for the production of SGMs via genetic experiments. Biochemical characterization demonstrated that the recombinant enzyme, SurE, can generate the main products SGM-A and -F from the corresponding SNAC substrates, indicating that SurE is a standalone thioesterase-like enzyme. SurE also displays considerable substrate plasticity with expanded ring or different amino acid compositions to produce different cyclopeptides, highlighting the potential of chemoenzymatic applications. Site-directed mutagenesis allowed identification of the key residues of SurE. Finally, bioinformatics analysis suggested that SurE homologs are widely distributed in bacteria, suggesting a general mechanism of NRP release in Nature.

Keywords: cyclase and hydrolase; non-ribosomal peptide biosynthesis; penicillin binding protein; surugamides.

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非核糖体肽（NRPs）在非核糖体肽合成酶（NRPS）复合物上生物合成，其中C端释放结构域通常卸载产物。有趣的是，surugamides（SGM）NRPS中不存在专门的释放结构域，该结构域指导环状八肽SGM-A至-E以及线性十肽SGM-F的生物合成。在此，我们通过遗传实验确认了surE对于SGMs生产的必要性。生化特征表明，重组酶SurE可以从相应的SNAC底物生成主要产物SGM-A和-F，这表明SurE是一种独立的硫酯酶样酶。SurE还显示出相当的底物可塑性，可以扩展环或不同的氨基酸组成来产生不同的环肽，突显了其在化学酶应用中的潜力。定点突变使我们能够识别SurE的关键残基。最后，生物信息学分析表明，SurE同源物在细菌中广泛分布，这表明自然界中NRP释放的一种普遍机制。