Carbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonhaem iron oxygenases (ROs), catalyses the regioselective and stereoselective dihydroxylation of carbazole and acts as a primary catalyst in the degradation of carbazole by various bacteria. CARDO consists of three components: a terminal oxygenase (Oxy; encoded by the carAa
gene), a ferredoxin (Fd; encoded by the carAc
gene) and a ferredoxin reductase (Red; encoded by the carAd
sp. KA1 possesses the carbazole-degradative plasmid pCAR3 (Shintani et al.
), which contains two CARDO genes (carAaICarAcI
ROs can be divided into five subgroups (IA, IB, IIA, IIB and III) based on their number of constituents and the nature of their redox centre (Batie et al.
). There are three types of RO reductases: class IA reductases contain FMN and a plant-type [2Fe–2S] cluster; class IB and III reductases contain FAD and a plant-type [2Fe–2S] cluster; and class IIA and IIB reductases contain FAD as their cofactor. Class IIA and IIB reductases are grouped into the bacterial oxygenase-coupled NADH-ferredoxin reductases (ONFRs). ONFRs are members of the glutathione reductase-type ferredoxin-NADP+
oxidoreductases (GR-type FNRs). Aliverti et al.
) have written an excellent review on GR-type FNRs.
The CARDOs of KA1 are classified as class IIA ROs. Other well studied CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3 and Nocardioides aromaticivorans IC177 have been grouped into classes III, III and IIB, respectively. Therefore, the CARDO system has two different types of Reds, although high primary sequence similarity exists in the Oxy (>45% identity and >70% similarity) between the three classes, as well as a similarity in the reactions catalysed.
Recently, we have reported several crystal structures of the components of CARDOs: FdIII
from P. resinovorans
CA10 (Nam et al.
sp. J3 (Nojiri et al.
), their electron-transfer complex (Ashikawa et al.
) and OxyIIB
from N. aromaticivorans
(Inoue et al.
). We have also crystallized RedIII
sp. J3 (Ashikawa et al.
) and FdIIA
sp. KA1 (Umeda et al.
). To date, several structures of RO reductases and related reductases have been determined: phthalate dioxygenase reductase (class IA; Correll et al.
), benzoate dioxygenase reductase (class IB; Karlsson et al.
), biphenyl dioxygenase reductase (class IIB; 36.5% sequence identity to RedIIA
; Senda et al.
), toluene dioxygenase reductase (class IIB; 30.2% sequence identity to RedIIA
; Friemann et al.
) and putidaredoxin reductase (41.3% sequence identity to RedIIA
; Sevrioukova et al.
). However, no structure of a class IIA RO reductase has been reported. In this paper, we describe the crystallization and preliminary X-ray diffraction study of RedIIA
(encoded by fdrII
, 420 amino-acid residues, 44.4 kDa molecular mass). The three-dimensional structure of class IIA Red will provide additional insight into the mechanisms of electron transfer and recognition of a suitable redox partner between RO components.