Nonribosomal biosynthesis of backbone-modified peptides
Nonribosomal biosynthesis of backbone-modified peptides
复制标题
DOI：
10.1038/nchem.2891
复制
发表时间：
2018-03-01
期刊：
影响因子：
21.8
通讯作者：
Hilvert, Donald
中科院分区：
文献类型：
--
作者：
Niquille, David L.;Hansen, Douglas A.;Hilvert, Donald
非核糖体肽骨架的生物合成修饰代表了调节一类重要治疗药物的结构和性质的潜在有力策略。使用催化活性的高通量测定，我们在这里表明，原型非核糖体肽合成酶的L-Phe特异性模块可以被重编程以接受和处理具有接近天然特异性和效率的骨架修饰的氨基酸（S）-β-Phe。与不可水解的氨酰-AMP类似物的共晶体结构揭示了40，000倍α/β特异性开关的起源，阐明了活性位点的微妙但精确的重塑。当工程化催化剂与下游模块配对时，在体外以制备规模（类似于1 mmol）和在体内以高滴度（类似于100 mg l（-1））产生含（S）-β-Phe的肽，突出了生物合成途径工程化用于构建新型非核糖体β-框架的潜力。
Biosynthetic modification of nonribosomal peptide backbones represents a potentially powerful strategy to modulate the structure and properties of an important class of therapeutics. Using a high-throughput assay for catalytic activity, we show here that an L-Phe-specific module of an archetypal nonribosomal peptide synthetase can be reprogrammed to accept and process the backbone-modified amino acid (S)-beta-Phe with near-native specificity and efficiency. A co-crystal structure with a non-hydrolysable aminoacyl-AMP analogue reveals the origins of the 40,000-fold alpha/beta-specificity switch, illuminating subtle but precise remodelling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-beta-Phe-containing peptides were produced at preparative scale in vitro (similar to 1 mmol) and high titres in vivo (similar to 100 mg l(-1)), highlighting the potential of biosynthetic pathway engineering for the construction of novel nonribosomal beta-frameworks.