将α-N-甲基化的非蛋白质氨基酸引入肽中可以提高其生物活性,膜通透性和蛋白水解稳定性。这通常是实现的,在自然界和实验室里,通过组装预甲基化的氨基酸。使酰胺键甲基化的更有吸引力的途径是具有挑战性的。生物学已经进化出一种α-N-自甲基化酶,OphMA,作用于与其C末端融合的肽的酰胺键。由于其底物的核糖体生物合成,这种酶对具有非蛋白氨基酸的肽的活性尚未得到解决。一个工程的OphMA,内含肽介导的蛋白质连接和固相肽合成使我们能够在非天然酰胺的情况下证明酰胺键的甲基化。该方法可应用于治疗性肽的生物技术生产。
肽骨架甲基化是非常需要的修饰。使用拆分intein,合成肽和甲基化酶的工程变体,对于OphMA2,可以酶促甲基化涉及非天然氨基酸的酰胺键。这在治疗性肽的生物技术生产中具有应用。
Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.
Peptide backbone methylation is a highly desired modification. Using split intein, synthetic peptides and an engineered variant of the methylase, fOphMA2, it was possible to enzymatically methylate amide bonds involving non‐natural amino acids. This has application in the biotechnological production of therapeutic peptides.