Dipeptide formation on engineered hybrid peptide synthetases
Dipeptide formation on engineered hybrid peptide synthetases
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DOI：
10.1016/s1074-5521(00)00118-6
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发表时间：
2000-06-01
影响因子：
--
通讯作者：
Marahiel, MA
中科院分区：
文献类型：
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作者：
Doekel, S;Marahiel, MA
背景：非核糖体肽合成酶（NRPSs）是一种模块化的“巨型酶”，它利用多载体硫模板机制催化大量生物活性肽的组装。这些模块包含特定的结构域，这些结构域作为不同的单元来催化与底物激活、修饰和缩合相关的特定反应。这种生物合成模板的排列引起了人们对工程新颖NRPSs的兴趣。结果：我们描述了一套双模块混合NRPSs的设计和构造。通过引入腺苷化和巯基化（PCP）结构域之间的融合，我们设计了合成二肽的模板。通过构建模板的特异性和腺苷酸化结构域的排列来定义预测的二肽，并在体外合成。通过测定底物腺苷化和硫基化的动力学参数，研究了分子内融合的影响。人工NRPSs上二肽的形成速率与天然模板相似。结论：可以推断出NRPSs对域交换耐受性的几个新方面。通过选择融合位点在腺苷化和PCP结构域的边界区域，我们发现PCP结构域没有一般的底物选择性。没有迹象表明缩合反应的选择性偏向于供体氨基酸，而在受体位置有一个大小决定的选择。此外，我们证明了天然延伸模块可以转化为肽键形成的起始模块。这些结果代表了在工程NRPSs上合理从头合成小肽的第一个例子。
Background: Nonribosomal peptide synthetases (NRPSs) are modular 'megaenzymes' that catalyze the assembly of a large number of bioactive peptides using the multiple carrier thiotemplate mechanism. The modules comprise specific domains that act as distinct units to catalyze specific reactions associated with substrate activation, modification and condensation. Such an arrangement of biosynthetic templates has evoked interest in engineering novel NRPSs.Results: We describe the design and construction of a set of dimodular hybrid NRPSs. By introducing domain fusions between adenylation and thiolation (PCP) domains we designed synthetic templates for dipeptide formation. The predicted dipeptides, as defined by the specificity and arrangement of the adenylation domains of the constructed templates, were synthesized in vitro. The effect of the intramolecular fusion was investigated by determining kinetic parameters for substrate adenylation and thiolation. The rate of dipeptide formation on the artificial NRPSs is similar to that of natural templates.Conclusions: Several new aspects concerning the tolerance of NRPSs to domain swaps can be deduced. By choosing the fusion site in the border region of adenylation and PCP domains we showed that the PCP domain exhibits no general substrate selectivity. There was no suggestion that selectivity of the condensation reaction was biased towards the donor amino acid, whereas at the acceptor position there was a size-determined selection. In addition, we demonstrated that a native elongation module can be converted to an initiation module for peptide-bond formation. These results represent the first example of rational de novo synthesis of small peptides on engineered NRPSs.