Senda T, Yamada T, Sakurai N, Kubota M, Nishizaki T, Masai E, Fukuda M, Mitsuidagger Y
Division of Protein Engineering, University of Technology, Nagaoka, Niigata, Japan.
J Mol Biol. 2000 Dec 1;304(3):397-410. doi: 10.1006/jmbi.2000.4200.
Oxidative biodegradation of aromatic compounds by bacteria usually begins with hydroxylation of the aromatic ring by multi-component dioxygenases like benzene dioxygenase, biphenyl dioxygenase, and others. These enzymes are composed of ferredoxin reductase, ferredoxin, and terminal oxygenase. Reducing equivalents that originate from NADH are transferred from ferredoxin reductase to ferredoxin and, in turn, to the terminal oxygenase, thus resulting in the activation of a dioxygen. BphA4 is the ferredoxin reductase component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102. The amino acid sequence of BphA4 exhibits significant homology with the putidaredoxin reductase of the cytochrome P450cam system in Pseudomonas putida, as well as with various other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) of bacteria. To date, no structural information has been provided for the ferredoxin reductase component of the dioxygenase systems. In order to provide a structural basis for discussing the mechanism of electron transport between ferredoxin reductase and ferredoxin, crystal structures of BphA4 and its NADH complex were solved. The three-dimensional structure of BphA4 is different from those of ferredoxin reductases whose structures have already been determined, but adopts essentially the same fold as the enzymes of the glutathione reductase (GR) family. Also the three-dimensional structure of the first two domains of BphA4 adopts a fold similar to that of adrenodoxin reductase (AdR) in the mitochondrial cytochrome P450 system. Comparing the amino acid sequence with what is known of the three-dimensional structure of BphA4 strongly suggests that the other ONFRs have secondary structural features that are similar to that of BphA4. This analysis of the crystal structures of BphA4 suggests that Lys53 and Glu159 seem to be involved in the hydride transfer from NADH to FAD. Since the amino acid residues around the active site, some of which seem to be important to electron transport, are highly conserved among ONFRs, it is likely that the mechanism of electron transport of BphA4 is quite applicable to other ONFRs.
细菌对芳香族化合物的氧化生物降解通常始于多组分双加氧酶（如苯双加氧酶、联苯双加氧酶等）对芳香环的羟基化作用。这些酶由铁氧化还原蛋白还原酶、铁氧化还原蛋白和末端加氧酶组成。源自 NADH 的还原当量从铁氧化还原蛋白还原酶转移到铁氧化还原蛋白，进而转移到末端加氧酶，从而导致双加氧的激活。BphA4 是来自假单胞菌属菌株 KKS102 的联苯双加氧酶的铁氧化还原蛋白还原酶组分。BphA4 的氨基酸序列与恶臭假单胞菌中细胞色素 P450cam 系统的恶臭假单胞菌铁氧化还原蛋白还原酶以及细菌的各种其他加氧酶偶联的 NADH 依赖性铁氧化还原蛋白还原酶（ONFRs）具有显著同源性。迄今为止，尚未提供双加氧酶系统中铁氧化还原蛋白还原酶组分的结构信息。为了为讨论铁氧化还原蛋白还原酶与铁氧化还原蛋白之间的电子传递机制提供结构基础，解析了 BphA4 及其 NADH 复合物的晶体结构。BphA4 的三维结构与已确定结构的铁氧化还原蛋白还原酶不同，但与谷胱甘肽还原酶（GR）家族的酶具有基本相同的折叠方式。此外，BphA4 前两个结构域的三维结构与线粒体细胞色素 P450 系统中的肾上腺皮质铁氧化还原蛋白还原酶（AdR）具有相似的折叠方式。将 BphA4 的氨基酸序列与其已知的三维结构进行比较强烈表明，其他 ONFRs 具有与 BphA4 相似的二级结构特征。对 BphA4 晶体结构的分析表明，Lys53 和 Glu159 似乎参与了从 NADH 到 FAD 的氢化物转移。由于活性位点周围的氨基酸残基（其中一些似乎对电子传递很重要）在 ONFRs 中高度保守，因此 BphA4 的电子传递机制很可能适用于其他 ONFRs。
