Rational manipulation of carrier-domain geometry in nonribosomal peptide synthetases
Rational manipulation of carrier-domain geometry in nonribosomal peptide synthetases
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DOI：
10.1002/cbic.200700010
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发表时间：
2007-04-16
期刊：
影响因子：
3.2
通讯作者：
Bruner, Steven D.
中科院分区：
文献类型：
--
作者：
Liu, Ye;Bruner, Steven D.
非核糖体肽合成酶(NRPS)和聚酮合成酶(PKS)负责一系列具有重要治疗意义的天然产物的生物合成。[1]NRPS和PKS各自由独立的折叠结构域组成，以线性流水线方式排列，组装的蛋白质序列决定了聚合物产品的同一性。NRPS和PKS途径的独特、合理的逻辑激发了使用基因工程操纵系统来生产分子库或具有所需性质的特定分子的提议。[2]单体构建块和不断增长的聚合物链共价连接到专用载体结构域，如磷酸铁硫酸酯(方案1A)。随着聚合物伸长的发生，不断增长的聚合物链在区域活性中心之间转移。NRPS和PKS组装中的拴系底物运输机制和载体结构域结构在很大程度上仍不清楚。尽管最近已经解决了多结构域PKS片段和相关脂肪酸合成酶的X射线结构，但载体结构域及其连接的磷酸铁蛋白基臂的活性构象尚未确定。[3]当结构域在结构域活性中心之间移动磷酸铁蛋白底物时，载体结构域可能发生显著的构象变化。在这里，我们报告了一种方法来操纵NRPS组件的载体域几何结构，通过应用磷酸铁硫酸酯的异构性非水解性类似物。负责将一个生长的多肽链延长一个氨基酸的核心NRPS机制由三个结构域组成：缩合(C)、腺化(A)和肽基载体蛋白(PCP)。[4]A结构域负责选择特定的氨基酸并将其加载到PCP结构域的磷酸丙氨酸臂上。C结构域通过形成肽键将上游氨基酸与不断增长的链连接起来。NRPS装配线的末端反应是由一个专门的硫代酯酶(TE)结构域释放多肽产品，该结构域通常位于C-末端模块。尽管NRPS、PKS和脂肪酸合成酶/合成酶在化学上有明显的差异，但硫酯模板的方法对所有这三个系统都是共同的。此外，载体和硫酯酶结构域在NRPS、PKS和脂肪酸合成酶/合成酶/合成中具有结构同源性，并且存在几个整合这三类酶结构域的混合酶系统的例子。[5]结构受限的多域NRPS组装将有助于阐明合成酶的复杂结构和机制。这里所描述的策略如方案1所示。在多域NRPS组装中，可以使用磷酸二氢呋喃基硫酯类化合物来操纵载体结构域的几何形状。辅酶A(CoA)的类似物通过PPTase酶被负载到apo-NRPS蛋白上。该偶联物被设计成将抑制物部分或反应性基团输送到感兴趣的区域。通过使用这一策略，可以设计不同的络合物来将磷酸铁氨酸臂定向到催化相关构象中特定结构域的活性位置。PPTase表现出广泛的底物特异性，并已被用作将不同的CoA类似物装载到载体结构域上的通用工具。[6]使用CoA类似物作为结构和机械探针的价值已在广泛的[a]Y.Liu博士、波士顿学院尤金·F·默克特和…的SD Bruner教授化学系中得到证明
Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are responsible for the biosynthesis of a wide range of therapeutically important natural products.[1] NRPS and PKS are each composed of independently folded domains, arranged in a linear assembly-line manner, and the protein sequence of the assembly determines the identity of the polymer product. The unique, rational logic of NRPS and PKS pathways has inspired the proposal to use genetic engineering to manipulate systems to produce libraries of molecules or a specific molecule with desired properties.[2] The monomer building blocks and the growing polymer chain are covalently linked to dedicated carrier domains as phosphopantetheinyl thioesters (Scheme1A). The growing polymer chain is transferred between domain active sites as polymer elongation occurs. The mechanism of tethered-substrate trafficking and carrierdomain architecture in NRPS and PKS assemblies remain largely unknown. Although X-ray structures of multidomain PKS fragments and the related fatty acid synthases have been solved recently, the active conformation of the carrier domains and their attached phosphopantetheinyl arms has not been determined.[3] Significant conformational changes in carrier domains are likely to occur as the domains move phosphopantetheinyl substrates between domain active sites. Here we report an approach to manipulate the carrier-domain geometry of NRPS assemblies through the application of isosteric nonhydrolyzable analogues of phosphopantetheinyl thioesters. The core NRPS machinery responsible for extending a growing peptide chain by one amino acid consists of three domains: condensation (C), adenylation (A), and peptidyl carrier protein (PCP).[4] The A domain is responsible for selecting a specific amino acid and loading it onto the phosphopantetheine arm of the PCP domain. The C domain couples the upstream amino acid with the growing chain through peptidebond formation. The terminal reaction of NRPS assembly lines is the release of the peptide product by a dedicated thioesterase (TE) domain, which is normally positioned at the C-terminal module. Although there are obvious differences in the chemistry of NRPS, PKS, and fatty acid synthetases/synthases, the thioester-templated methodology is common to all three systems. In addition, carrier and thioesterase domains share structural homology among NRPS, PKS, and fatty acid synthetases/syntheses, and several examples of hybrid enzyme systems that integrate enzyme domains from the three classes exist.[5] Structurally restrained multidomain NRPS assemblies will be useful in elucidating the complex structure and mechanism of synthetases. The strategy described here is shown in Scheme 1. Phosphopantetheinyl thioester mimics can be used to manipulate the geometry of carrier domains in multidomain NRPS assemblies. Analogues of coenzyme A (CoA) are loaded onto apo-NRPS proteins by phosphopantetheinyl transferase (PPTase) enzymes. The conjugates are designed to deliver an inhibitor moiety or reactive group to a domain of interest. By using this strategy, various complexes can be designed to direct the phosphopantetheine arm to the active site of a specific domain in a catalytically relevant conformation. PPTases display broad substrate specificity and have been exploited as general tools to load diverse CoA analogues onto carrier domains.[6] The value of using analogues of CoA as structural and mechanistic probes has been demonstrated in a wide range of [a] Y. Liu, Prof. Dr. SD Bruner Department of Chemistry, Boston College Eugene F. Merkert …