Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase
Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase
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DOI：
10.1128/aem.04000-13
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发表时间：
2014-05-01
影响因子：
4.4
通讯作者：
Nojiri, Hideaki
中科院分区：
文献类型：
--
作者：
Inoue, Kengo;Usami, Yusuke;Nojiri, Hideaki
咔唑1，9 α-双加氧酶（Carbazole 1，9a-dioxygenase，CARDO）是一种Rieske型非血红素铁加氧酶（nonheme iron oxygenase，RO），由末端加氧酶（terminal oxygenase，Oxy）、铁氧还蛋白（ferredoxin）和铁氧还蛋白还原酶（ferredoxin reductase）组成。氧对咔唑具有角向双加氧活性。以前，来自Janthinobacterium sp.菌株J3的氧编码基因的定点诱变产生了I262 V、F275 W、Q282 N和Q282 Y氧衍生物，其显示出与野生型酶不同的氧合能力。为了理解导致不同氧化反应的结构特征，我们确定了衍生物的晶体结构，包括游离的和与底物络合的。I262 V、F275 W和Q282 Y衍生物催化咔唑的侧向二氧化，产率高于野生型。先前的研究确定了与咔唑络合的Oxy的晶体结构，并揭示了Gly 178的羰基氧与咔唑的亚氨基氮形成氢键。在这些衍生物中，与野生型相比，咔唑分别旋转约15，25和25，为水分子创造空间，水分子与Gly 178的羰基氧和咔唑的亚氨基氮形成氢键。在与芴络合的F275 W衍生物的晶体结构中，芴的C-9（其对应于咔唑的亚氨基氮）取向为接近突变的残基Trp 275，其位于Gly 178的羰基氧的结合口袋的相对侧。我们的结构分析表明，在RO的substrabinding口袋的表面上的疏水残基的微调引起轻微的位移的基板结合的位置，反过来，有利于特定的氧化反应对各种基板。
Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substratebinding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.