Insights into the mechanism of peptide cyclodehydrations achieved through the chemoenzymatic generation of amide derivatives.
Insights into the mechanism of peptide cyclodehydrations achieved through the chemoenzymatic generation of amide derivatives.
复制标题
DOI：
10.1021/ja4029507
复制
发表时间：
2013-06-12
影响因子：
15
通讯作者：
Mitchell, Douglas A.
中科院分区：
文献类型：
--
作者：
Dunbar, Kyle L.;Mitchell, Douglas A.
目前用于产生带有酰胺羰基衍生物的肽和蛋白质的策略依赖于用于酰胺官能化的固相肽合成。虽然这种策略已经成功实施，但技术限制限制了合成片段的长度和序列。在这里，我们报告了噻唑/恶唑修饰的小菌素（TOMM）cyclopropylase的再利用位点特异性地安装到不同的肽底物上的酰胺骨架标签，我们称之为唑啉介导的肽骨架标记（AMPL）的方法。这种方便的化学酶策略可以生成硫代酰胺和具有同位素标记的氧原子的酰胺。此外，我们证明了TOMM合成酶的第一个前导肽独立活性，避免了将感兴趣的序列融合到前导肽进行修饰的要求。通过生物信息学指导的定点突变，我们还将严格依赖于酶的TOMM唑合成酶转化为唑啉合成酶。这通过允许使用任何体外重构的TOMM合成酶而极大地扩展了可由AMPL修饰的底物的谱。为了证明AMPL的实用性的机制酶学研究，18 O-标记的底物产生提供直接的证据表明，在TOERONE发生环化脱水通过磷酸化的羰基氧之前的环化残基。此外，我们表明，AMPL是一个有用的工具，用于建立在体外修饰的肽和含唑啉的天然产物的位置的唑啉。
Current strategies for generating peptides and proteins bearing amide carbonyl derivatives rely on solid-phase peptide synthesis for amide functionalization. Although such strategies have been successfully implemented, technical limitations restrict both the length and sequence of the synthetic fragments. Herein we report the repurposing of a thiazole/oxazole-modified microcin (TOMM) cyclodehydratase to site-specifically install amide backbone labels onto diverse peptide substrates, a method we refer to as azoline-mediated peptide backbone labeling (AMPL). This convenient chemoenzymatic strategy can generate both thioamides and amides with isotopically labeled oxygen atoms. Moreover, we demonstrate the first leader peptide-independent activity of a TOMM synthetase, circumventing the requirement that sequences of interest be fused to a leader peptide for modification. Through bioinformatics-guided site-directed mutagenesis, we also convert a strictly dehydrogenase-dependent TOMM azole synthetase into an azoline synthetase. This vastly expands the spectrum of substrates modifiable by AMPL by allowing any in vitro reconstituted TOMM synthetase to be employed. To demonstrate the utility of AMPL for mechanistic enzymology studies, an 18O-labeled substrate was generated to provide direct evidence that cyclodehydrations in TOMMs occur through the phosphorylation of the carbonyl oxygen preceding the cyclized residue. Furthermore, we demonstrate that AMPL is a useful tool for establishing the location of azolines both on in vitro modified peptides and azoline-containing natural products.
登录
查看更多内容
影响因子：
2.1
作者：
BROWN, DW;CAMPBELL, MM;WALKER, CV
通讯作者：
WALKER, CV
影响因子：
14.8
作者：
Donia, Mohamed S.;Hathaway, Brian J.;Schmidt, Eric W.
通讯作者：
Schmidt, Eric W.
影响因子：
15
作者：
Hu, J;Chekmenev, EY;Cross, TA
通讯作者：
Cross, TA
影响因子：
15
作者：
Levengood, Matthew R.;Patton, Gregory C.;van der Donk, Wilfred A.
通讯作者：
van der Donk, Wilfred A.
影响因子：
4.8
作者：
Gonzalez, David J.;Lee, Shaun W.;Dorrestein, Pieter C.
通讯作者：
Dorrestein, Pieter C.