Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism.
Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism.
复制标题
DOI：
10.1039/d2cb00005a
复制
发表时间：
2022-03-09
影响因子：
4.1
通讯作者：
Burkart MD
中科院分区：
文献类型：
--
作者：
Kim WE;Ishikawa F;Re RN;Suzuki T;Dohmae N;Kakeya H;Tanabe G;Burkart MD
非核糖体肽合成酶（NRPS）是一种复杂的多模块酶，含有催化结构域，负责将氨基酸装载和掺入天然产物中。这些独特的分子工厂可以产生具有非蛋白原性d-氨基酸的肽，其中差向异构化（E）结构域催化l-氨基酸转化为d-氨基酸，但其机制仍不完全清楚。在这里，我们描述了泛酰巯基乙胺交联探针的发展，模仿天然底物的酪氨酸合成酶，TycA的起始模块的L-苯丙氨酸，阐明和研究的E域的催化残基。基于机理的交联测定和MALDI-TOF MS用于鉴定H743和E882作为交联位点残基，证明它们作为催化碱的作用。诱变研究进一步验证了这些结果，并允许比较催化残基之间的反应性，得出的结论是谷氨酸盐在交联反应中充当主要亲核试剂，类似于差向异构化反应中氨基酸Cα-H的去质子化。这些研究中采用的交联探针为研究E结构域的分子细节以及研究C结构域的潜力提供了新的工具。特别是，他们将阐明这些结构域如何在自然界中发挥作用并与其底物相互作用的关键信息，进一步增强帮助NRPS系统组合生物合成努力产生新化合物所需的知识。使用含有靶向结构域的催化残基（His和Glu）的磺酰基弹头的泛酰巯基乙胺类似物交联探针研究NRPS的起始模块中的差向异构化（E）结构域机制。
Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the Cα-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds. Epimerization (E) domain mechanism in initiaion modules of NRPSs studied using pantetheine analog crosslinking probes containing sulfonyl warheads that target domain's catalytic residues (His and Glu).
登录
查看更多内容
影响因子：
16.6
作者：
Konno, Sho;La Clair, James J.;Burkart, Michael D.
通讯作者：
Burkart, Michael D.
影响因子：
11.9
作者：
Hur GH;Vickery CR;Burkart MD
通讯作者：
Burkart MD
影响因子：
3.2
作者：
Worthington, AS;Burkart, MD
通讯作者：
Burkart, MD
影响因子：
14.8
作者：
Weerapana, Eranthie;Simon, Gabriel M.;Cravatt, Benjamin F.
通讯作者：
Cravatt, Benjamin F.
DOI：
10.1039/c4cc09412c
发表时间：
2015-02-11
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Konno S;Ishikawa F;Suzuki T;Dohmae N;Burkart MD;Kakeya H
通讯作者：
Kakeya H