Nam Jeong-Won, Nojiri Hideaki, Noguchi Haruko, Uchimura Hiromasa, Yoshida Takako, Habe Hiroshi, Yamane Hisakazu, Omori Toshio
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Japan.
Appl Environ Microbiol. 2002 Dec;68(12):5882-90. doi: 10.1128/AEM.68.12.5882-5890.2002.
The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the k(cat)/K(m) for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.
树脂糖假单胞菌CA10菌株的咔唑1,9a -双加氧酶（CARDO）系统由末端加氧酶（CarAa）、铁氧化还原蛋白（CarAc）和铁氧化还原蛋白还原酶（CarAd）组成。CARDO的每个组分都在大肠杆菌BL21(DE3)菌株中以天然形式（CarAa）或His标签形式（CarAc和CarAd）表达，并纯化至表观均一性。发现CarAa是三聚体，每个单体中有一个Rieske型[2Fe - 2S]簇和一个单核铁中心。发现两种His标签蛋白均为单体，并含有从推导的氨基酸序列预测的辅基（His标签的CarAd，每个单体蛋白一个FAD和一个[2Fe - 2S]簇；His标签的CarAc，每个单体蛋白一个Rieske型[2Fe - 2S]簇）。NADH和NADPH均作为His标签的CarAd的有效电子供体。然而，由于在2,6 -二氯酚靛酚还原酶测定中NADH的k(cat)/K(m)比NADPH高22.3倍，因此NADH被认为是CarAd的生理电子供体。在存在NADH的情况下，His标签的CarAc被His标签的CarAd还原。同样，CarAa被His标签的CarAc、His标签的CarAd和NADH还原。这三种纯化的蛋白可以在体外重建CARDO活性。在重建的CARDO系统中，His标签的CarAc似乎是电子传递所必需的，而His标签的CarAd可以被一些无关的还原酶替代。
