Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code
Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code
复制标题
定向进化揭示腺苷酸化域特异性代码的功能序列空间
DOI：
10.1021/acschembio.9b00532
复制
发表时间：
2019
影响因子：
4
通讯作者：
Thomas, Michael George
中科院分区：
文献类型：
--
作者：
Throckmorton, Kurt;Vinnik, Vladimir;Chowdhury, Ratul;Cook, Taylor;Chevrette, Marc G.;Maranas, Costas;Pfleger, Brian;Thomas, Michael George
非核糖体肽是由非核糖体肽合成酶(NRPS)生物合成的重要天然产物。NRPS的腺化(A)结构域对于它们识别的底物具有高度的特异性。这种识别是由底物结合口袋中的10个残基决定的，称为特异码。这一发现导致了非核糖体多肽可以通过特异性密码交换来改变的建议。不幸的是，这种方法已经被证明是无效的，除了极少数例外；改变特异性代码通常会导致特异性扩大或功能不佳。为了加强我们对A结构域底物选择性的理解，我们对Entf的A结构域的特异性编码进行了详细的分析，Entf是一种参与大肠杆菌中肠杆菌素生物合成的NRPS。使用定向进化和遗传选择，我们确定了密码中哪些位置有严格的残基要求，哪些位置耐变异。我们证明了Entf A结构域和其他l-Ser特异的A结构域具有用于1-Ser识别的功能序列空间，而不是单一的编码。这个功能空间比所有表征的l-Ser特异性A结构域的集合更具扩展性：我们识别了152个新的l-Ser特异性编码。总而言之，我们的数据为如何克服阻碍A域专用性合理更改的障碍提供了重要的见解。
Nonribosomal peptides are important natural products biosynthesized by nonribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs are highly specific for the substrate they recognize. This recognition is determined by 10 residues in the substrate-binding pocket, termed the specificity code. This finding led to the proposal that nonribosomal peptides could be altered by specificity code swapping. Unfortunately, this approach has proven, with few exceptions, to be unproductive; changing the specificity code typically results in broadened specificity or poor function. To enhance our understanding of A domain substrate selectivity, we carried out a detailed analysis of the specificity code from the A domain of EntF, an NRPS involved in enterobactin biosynthesis inEscherichia coli. Using directed evolution and a genetic selection, we determined which sites in the code have strict residue requirements and which are tolerant of variation. We showed that the EntF A domain, and otherl-Ser-specific A domains, have a functional sequence space forl-Ser recognition, rather than a single code. This functional space is more expansive than the aggregate of all characterizedl-Ser-specific A domains: we identified 152 newl-Ser specificity codes. Together, our data provide essential insights into how to overcome the barriers that prevent rational changes to A domain specificity.