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Structural Basis of the Substrate Specificity of Rieske Nonheme Iron Oxygenase

Rieske 非血红素铁加氧酶底物特异性的结构基础

基本信息

批准号：
17380052
负责人：
NOJIRI Hideaki
金额：
$ 10.14万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

Carbazole 1,9a-dioxygenase (CARDO) catalyzes angular dioxygenation (AD) for carbazole (at 1, 9a carbons), lateral dioxygenation (LD) for polycyclic aromatic hydrocarbons and biphenyl, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To clarify the molecular mechanism determining the substrate specificity of this novel enzyme, 28 site-specific mutants having single (17) or double (11) amino acid substitution (s) of I262, F275, Q282, and/or F329, which located close vicinity to active site iron in substrate-binding pocket, were created. Then, their oxygenation activities for various aromatic compounds were determined. In addition, some of the mutant enzymes showing markedly different substrate specificity than wild-type CARDO were crystallized and their three-dimensional structures were determined as single and/or substrate-bound forms.As the results, it was revealed that I262 and F275 were important amino acid residues, which largely affected the substrate binding manner of carbazole, fluorene, and/or fluoranthene. Structural analyses clearly indicated that these amino acid substitutions resulted in the change in the docking manners of the some of the substrates. Different docking positions lead different carbon atoms of substrates to situate closer to active site iron than the case with wild type enzyme. This is a reason why CARDO mutants catalyze the different type of oxygenation reactions.

咔唑1，9 α-双加氧酶（CARDO）催化咔唑（在1，9 α碳上）的角向双加氧（AD）、多环芳烃和联苯的侧向双加氧（LD）以及芴的亚甲基碳和二苯并噻吩的硫化物硫的单加氧。为了阐明决定这种新型酶的底物特异性的分子机制，产生了28个位点特异性突变体，其具有I262、F275、Q282和/或F329的单（17）或双（11）氨基酸取代，其位于底物结合口袋中的活性位点铁附近。然后，测定了它们对各种芳香族化合物的氧化活性。此外，对底物特异性与野生型CARDO有显著差异的突变酶进行了结晶，并以单一和/或底物结合形式测定了它们的三维结构，结果显示I262和F275是重要的氨基酸残基，它们对咔唑、芴和/或荧蒽的底物结合方式有很大影响。结构分析清楚地表明，这些氨基酸取代导致了一些底物的对接方式的变化。不同的对接位置导致底物的不同碳原子比野生型酶更接近活性位点铁。这就是为什么CARDO突变体催化不同类型的氧化反应的原因。