Rieske nonhaem iron oxygenase systems (ROSs) are the initial catalysts in the degradation pathways of various aromatic compounds, including dioxins, polychlorinated biphenyls and crude-oil components such as polycyclic aromatic hydrocarbons and heteroaromatics, which are of serious environmental concern (Wittich, 1998
; Bressler & Fedorak, 2000
; Nojiri & Omori, 2002
; Habe & Omori, 2003
; Furukawa et al.
). With very few exceptions, ROSs catalyze the incorporation of both O atoms of molecular dioxygen at tandem C atoms of an aromatic ring as two hydroxyl groups in the cis
configuration. This dioxygenation is known as lateral dioxygenation or cis
-dihydroxylation (Mason & Cammack, 1992
). On the other hand, in the initial dioxygenation of several heteroaromatics such as carbazole and dioxins one carbon that is bonded to the heteroatom and its adjacent carbon in the aromatic ring are both hydroxylated. This reaction, called angular dioxygenation, is catalyzed by a limited number of ROSs, which are called angular dioxygenases (Nojiri & Omori, 2002
). The terminal oxygenase components of ROSs invariably consist of an iron–sulfur protein with a Rieske-type [2Fe–2S] cluster and a mononuclear iron. The electron-transport chain consists of either one or two separate proteins: reductase alone or ferredoxin and reductase in combination (Mason & Cammack, 1992
). ROSs catalyze the dioxygenation of aromatics using dioxygen and two electrons. The electrons, which are originally derived from NAD(P)H, are transferred through the electron-transport proteins. ROSs have been classified into three major groups based on the number of constituent components and the nature of the redox centre (Batie et al.
; Ferraro et al.
). Class I ROSs consist of reductase and oxygenase components, with the reductases containing both flavin and a plant-type [2Fe–2S] cluster, and are further divided into two subclasses by the type of flavin, FMN (class IA) or FAD (class IB). Both class II and III ROSs contain a ferredoxin component in addition to the reductase and oxygenase components. Class II is further divided into classes IIA and IIB, which use putidaredoxin-type and Rieske-type ferredoxins, respectively, to mediate electron transfer between the reductase and oxygenase. Class II reductases contain FAD as the only cofactor, whereas class III reductases contain FAD and a plant-type [2Fe–2S] cluster.
We have investigated the enzymatic function of carbazole 1,9a-dioxygenase (CARDO) from various bacteria (Nojiri & Omori, 2002
; Inoue et al.
). All CARDOs consist of three components: a terminal oxygenase CARDO-O, a ferredoxin CARDO-F and a ferredoxin reductase CARDO-R, which are encoded by carAa
) and carAd
) genes, respectively (Fig. 1). The CARDOs from Pseudomonas resinovorans
sp. J3 (CARDOJ3
sp. KA1 (CARDOKA1
) and Nocardioides aromaticivorans
) are grouped into classes III, III, IIA and IIB, respectively (Sato et al.
; Inoue et al.
; Urata et al.
), indicating that these CARDOs have diverse types of electron-transfer components (CARDO-F and CARDO-R). Therefore, CARDO is an excellent model system for studying structure–function relationships and the mechanisms of inter-component electron transfer in ROSs.
Figure 1 Components, functions and structures solved for the clas III CARDO system. The proposed electron-transfer reactions and the conversion of carbazole to 2′-aminobiphenyl-2,3-diol are illustrated. The subscripts ‘ox’ and ‘red’ (more ...)
Recently, we determined the crystal structures of CARDO-F from P. resinovorans
; Nam et al.
), CARDO-O from Janthinobacterium
sp. J3 (CARDO-OJ3
; Nojiri et al.
; Fig. 1) and the electron-transfer complex between CARDO-OJ3
(Ashikawa et al.
). Class III ROS reductases, including CARDO-RCA10
, contain a plant-type [2Fe–2S] cluster and FAD and their structures have not yet been determined. ROS reductases are classified into two separate structural families: ferredoxin-NADP reductases (FNR) and glutathione reductases (GR). The structures of two FNR-family ROS reductases, phthalate dioxygenase reductase (PDO-R; class IA) from P. cepacia
PHK (Correll et al.
) and benzoate dioxygenase reductase (BZDO-R; class IB) from Acinetobacter baylyi
ADP1 (Karlsson et al.
), have been determined. The structure of biphenyl dioxygenase reductase from Pseudomonas
sp. KKS102, which belongs to the GR family, has also been determined (class IIB; Senda et al.
). Although the amino-acid sequences of class III CARDO-Rs appear to align well with that of BZDO-R (23–24% amino-acid sequence identity), there may be significant structural differences between class III CARDO-Rs and BZDO-R as the reductases have different redox partners (a ferredoxin component and an oxygenase component, respectively). Structural and functional studies on class III CARDO-Rs could enrich our knowledge and further our understanding of the mechanisms of electron transfer between ROS components. For this purpose, we report the crystallization of and preliminary X-ray diffraction studies on a class III CARDO-R (both CARDO-RCA10
were composed of 329 amino acids with a molecular weight of approximately 36 kDa).