Structural Basis of the Substrate Specificity of Rieske Nonheme Iron Oxygenase

咔唑1,9a-双加氧酶（CARDO）催化咔唑（1,9a碳）的角双氧合（AD），多环芳烃和联苯的侧向双氧合（LD），芴的亚甲基碳和二苯并噻吩的硫的单氧合。为了阐明决定这种新酶底物特异性的分子机制，我们创建了28个位点特异性突变体，它们位于底物结合口袋中靠近活性位点铁的I262、F275、Q282和/或F329的单（17）或双（11）个氨基酸取代。然后测定了它们对各种芳香族化合物的氧化活性。此外，一些与野生型CARDO表现出明显不同底物特异性的突变酶被结晶，它们的三维结构被确定为单一和/或底物结合形式。结果表明，I262和F275是重要的氨基酸残基，它们在很大程度上影响了咔唑、芴和/或氟蒽的底物结合方式。结构分析清楚地表明，这些氨基酸取代导致了一些底物对接方式的改变。不同的对接位置导致底物的不同碳原子比野生型酶更靠近活性位点铁。这就是为什么CARDO突变体催化不同类型的氧化反应的原因。

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.