Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.
Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.
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DOI：
10.1371/journal.pone.0138434
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Watanabe Y
中科院分区：
文献类型：
--
作者：
Watanabe S;Sueda R;Fukumori F;Watanabe Y
冠瘿碱，特别是胭脂碱和章鱼碱，是在由农杆菌属物种感染诱导的冠瘿肿瘤组织中发现的特异性化合物，并且由充分研究的NAD（P）H依赖性脱氢酶（脱氢酶）合成，所述脱氢酶催化α-酮戊二酸或丙酮酸与L-精氨酸的还原缩合。相应的基因通过肿瘤诱导（Ti）质粒转移到植物细胞中。除了逆氧化反应之外，基因noxB-noxA和ooxB-ooxA被认为参与冠冠瘿碱（膜相关）氧化酶;然而，由于与纯化（和保存）相关的困难，它们的性质尚未详细阐明。本文中，我们成功地在恶臭假单胞菌细胞中表达了来自恶臭假单胞菌的Nox/Oox样基因。纯化的蛋白质由OdhA、OdhB和OdhC基因编码的不同的α-、β-和γ-亚基组成，它们串联排列在染色体上（OdhB-C-A），并且在人工电子受体如2，6-二氯靛酚存在下对胭脂碱表现出脱氢酶（但不表现出氧化酶）活性。该酶含有FAD、FMN和[2Fe-2S]-铁硫作为辅基。而慢生型大豆根瘤菌的基因簇由Odh B 1 -C-A-B 2组成，其中每个β亚基与共同的α和γ亚基组装形成两个蛋白质Odh AB 1C和Odh AB 2C。尽管OdhB 1和OdhB 2都具有章鱼碱降解酶的功能，但它们之间的系统发育关系并不密切，这为通过“亚基交换”获得新的功能提供了明确的证据。据我们所知，这是第一个研究含有黄素的冠瘿碱脱氢酶。
Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB 1 -C-A-B 2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.