Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase.
Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase.
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DOI：
10.1074/jbc.ra120.015932
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Drennan CL
中科院分区：
文献类型：
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作者：
Jonnalagadda R;Del Rio Flores A;Cai W;Mehmood R;Narayanamoorthy M;Ren C;Zaragoza JPT;Kulik HJ;Zhang W;Drennan CL
异腈部分存在于海绵和一些微生物中，在毒力和金属获取等过程中发挥作用。直到最近，对于异腈生物合成，仅一种途径是已知的，这是将I-Trp/I-Tyr的氮原子与来自核酮糖-5-磷酸的碳原子结合在一起的缩合反应。随着ScoE的发现，一种来自天蓝淡红链霉菌的单核Fe（II）α-酮戊二酸依赖性双加氧酶，确定了第二条途径。ScoE从甘氨酸加合物形成异腈，其中氮和碳原子都来自相同的甘氨酰基部分。该反应是异腈脂肽的非核糖体生物合成途径的一部分。在这里，我们提出了结构，生物化学和计算研究的机制，异腈形成的斯科E，前所未有的反应，单核铁（II）α-酮戊二酸依赖性双加氧酶超家族。测量了该酶促反应的化学计量，并呈现了Fe（II）结合的ScoE的多个高分辨率（1.45-1.96 μ m分辨率）晶体结构，提供了对底物（R）-3-（（羧甲基）氨基）丁酸（CABA）、共底物α-酮戊二酸和Fe（IV）=O模拟氧钒的结合的深入了解。与先前发表的ScoE晶体结构的比较表明，ScoE有一个“诱导型”α-酮戊二酸结合位点，其中两个残基精氨酸-157和组氨酸-299从蛋白质表面移动约10 μ m进入活性位点，产生一个瞬时α-酮戊二酸结合口袋。总之，来自结构分析、定点诱变和计算的数据提供了对α-酮戊二酸结合模式、异腈形成机制以及ScoE的结构如何适应于进行这种不寻常的化学反应的深入了解。
The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45–1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an “inducible” α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.