Lee Jungkul, Simurdiak Michael, Zhao Huimin
Departments of Chemical and Biomolecular Engineering and Chemistry, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 2005 Nov 4;280(44):36719-27. doi: 10.1074/jbc.M505334200. Epub 2005 Sep 2.
Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.
铁硫蛋白氧化酶催化各种各样重要的氧化反应。在此，我们报道了一种新型铁硫蛋白N - 氧化酶——氨基吡咯硝菌素氧化酶（PrnD）的特性，该酶催化芳胺向芳基硝基的异常氧化反应。荧光假单胞菌Pf5的PrnD在大肠杆菌中实现了功能表达，并对纯化后的PrnD的活性进行了重构，这需要在体外将铁硫蛋白铁硫簇装配到蛋白质中，并且需要烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、黄素单核苷酸（FMN）和大肠杆菌黄素还原酶SsuE的存在。生化和生物信息学研究表明，重构后的PrnD包含一个铁硫蛋白铁硫簇和一个单核铁中心，它们分别由半胱氨酸（Cys）（69）、半胱氨酸（88）、组氨酸（His）（71）、组氨酸（91）、天冬氨酸（Asp）（323）、组氨酸（186）和组氨酸（191）的残基形成。该酶显示出有限的底物特异性范围，并催化氨基吡咯硝菌素转化为吡咯硝菌素，米氏常数（K(m)） = 191微摩尔，催化常数（k(cat)） = 6.8分钟-1。用（¹⁸）O₂和H₂（¹⁸）O进行的同位素标记实验表明，吡咯硝菌素产物中的氧原子仅来源于分子氧。此外，还发现PrnD催化芳胺底物的氧化反应发生在酶的活性位点，且不涉及自由基链反应。通过类比已知的芳胺氧化实例，提出了氨基吡咯硝菌素生物转化为吡咯硝菌素的催化机制。我们的结果应有助于对这种芳胺氧化酶进行进一步的机制和晶体学研究，并可能为从相应的芳香胺合成芳香硝基化合物提供一条新的酶促途径。
