An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket
An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket
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具有扩大底物结合口袋的工程芳基酸腺苷酸化结构域
DOI：
10.1002/anie.201900318
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
G.
中科院分区：
文献类型：
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作者：
Ishikawa;F.;Miyanaga;A.;Kitayama;H.;Nakamura;S.;Nakanishi;I.;Kudo;F.;Eguchi;T.;Tanabe;G.
腺苷酸化（A）结构域作为非核糖体肽合成酶（NRPS）的看门人，确保正确的氨基酸/芳基酸结构单元的活化和硫代酯化。芳基酸结构单元最常在铁螯合铁载体中观察到，但不限于它们。人们对芳香酸A结构域的重编程知之甚少。我们表明，一个单一的天冬酰胺-甘氨酸突变的芳基酸A域导致的酶，容忍范围广泛的非天然的芳基酸。工程化催化剂能够活化用硝基、氰基、溴和碘基团官能化的非天然芳基酸，即使没有观察到野生型酶对这些底物的酶活性。与不可水解的芳基AMP类似物的共晶体结构揭示了这种底物混杂扩展的起源，突出了酶的底物结合口袋的扩大。我们的研究结果可能被利用来生产多样化的含芳基酸的天然产物，并作为模板，在组合生物合成中进一步定向进化。
Adenylation (A) domains act as the gatekeepers of non‐ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron‐chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A‐domains. We show that a single asparagine‐to‐glycine mutation in an aryl acid A‐domain leads to an enzyme that tolerates a wide range of non‐native aryl acids. The engineered catalyst is capable of activating non‐native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild‐type enzyme was observed toward these substrates. Co‐crystal structures with non‐hydrolysable aryl‐AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.