Construction of hybrid peptide synthetases by module and domain fusions

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

非核糖体肽合成酶（Nonribosomal peptide synthetases）是一类模块化酶，可组装形成结构多样且具有重要生物活性的肽类化合物。其模块化架构为通过重组生物合成基因理性设计新型化合物提供了巨大潜力。本研究报道了基于全模块融合策略，将双模块体系拓展为三模块体系的研究工作。研究对重组杂合酶进行了纯化，以体外监测产物的组装过程。我们以短杆菌酪肽合成酶（tyrocidine synthetase）的前两个模块为起点构建此类杂合体系，这两个模块可催化二肽D-苯丙氨酸-脯氨酸（dPhe-Pro）的形成。将该酶的第二个脯氨酸特异性模块，分别与短杆菌酪肽合成酶的第九、第十模块（分别识别鸟氨酸与亮氨酸）进行融合，得到了兼具两种底物特异性的双模块杂合酶。研究中将硫酯酶结构域（thioesterase domain）融合至末端模块。将此类双模块酶与负责掺入D-苯丙氨酸的短杆菌酪肽合成酶首个模块TycA共孵育后，成功以0.15 min⁻¹和2.1 min⁻¹的速率获得了预期的三肽产物D-苯丙氨酸-脯氨酸-鸟氨酸（dPhe-Pro-Orn）与D-苯丙氨酸-脯氨酸-亮氨酸（dPhe-Pro-Leu）。内部硫酯酶结构域足以且必需从杂合酶中释放产物，从而推动催化循环的进行。本研究采用的全模块融合策略基于对融合位点的优化界定，可克服近期发现的内源缩合结构域（condensation domains）的编辑功能所带来的障碍。通过催化亚基逐步构建杂合肽合成酶的研究思路，进一步证实了该体系在合成新型定制肽类化合物方面的内在潜力。