An enhanced chemoenzymatic method for loading substrates onto carrier protein domains
An enhanced chemoenzymatic method for loading substrates onto carrier protein domains
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DOI：
10.1139/bcb-2017-0275
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发表时间：
2018-06-01
影响因子：
2.9
通讯作者：
Cryle, Max J.
中科院分区：
文献类型：
--
作者：
Kittilae, Tiia;Cryle, Max J.
非核糖体肽合成酶（NRPS）机器产生许多与医学相关的肽，这些肽不容易通过化学合成获得。因此，了解NRPS机制对于有效地重新设计这些机制以产生新化合物至关重要。在nrpps介导的合成过程中，底物共价附着在肽基载体蛋白（pcp）上，而生产装载底物的pcp的困难阻碍了nrpps的研究。将底物装载到PCP结构域的不同方法已经被描述过，但都存在化学合成复杂或酶效率低的困难。在这里，我们描述了一种增强的化学酶加载方法，将两种方法结合成一个单一的，高效的一锅加载反应。首先，D-pantetheine和ATP通过辅酶A （CoA）生物合成中的2种酶的作用转化为去磷酸辅酶A。接下来，Sfp突变体R4-4使用碱性磷酸酶对磷酸腺苷酸进行去磷酸化，使连接体附着在PCP结构域上，磷酸化腺苷酸被抑制。该途径不依赖于常见问题的去磷酸辅酶a激酶的活性，因此提供了一种将底物装载到PCP结构域的改进方法。
Non-ribosomal peptide synthetase (NRPS) machineries produce many medically relevant peptides that cannot be easily accessed by chemical synthesis. Thus, understanding NRPS mechanism is of crucial importance to allow efficient redesign of these machineries to produce new compounds. During NRPS-mediated synthesis, substrates are covalently attached to peptidyl carrier proteins (PCPs), and studies of NRPSs are impeded by difficulties in producing PCPs loaded with substrates. Different approaches to load substrates onto PCP domains have been described, but all suffer from difficulties in either the complexity of chemical synthesis or low enzymatic efficiency. Here, we describe an enhanced chemoenzymatic loading method that combines 2 approaches into a single, highly efficient one-pot loading reaction. First, D-pantetheine and ATP are converted into dephospho-coenzyme A via the actions of 2 enzymes from coenzyme A (CoA) biosynthesis. Next, phosphoadenylates are dephosphorylated using alkaline phosphatase to allow linker attachment to PCP domain by Sfp mutant R4-4, which is inhibited by phosphoadenylates. This route does not depend on activity of the commonly problematic dephospho-CoA kinase and, therefore, offers an improved method for substrate loading onto PCP domains.