Carbazole, a toxic and carcinogenic compound (Sverdrup et al.
; Jha & Bharti, 2002
), is a component of creosote, crude oil and shale oil (Mushrush et al.
). In order to investigate the biodegradation of this N-heterocyclic aromatic compound in contaminated soil, many carbazole-degrading bacteria have been isolated. From analyses of the metabolism of carbazole by several bacteria, genes encoding carbazole-degradative enzymes, called the car
gene cluster, have been found. Subsequent enzymatic investigation revealed the importance of carbazole 1,9a-dioxygenase (CARDO) as the initial catalyst in the carbazole-degradation pathway (Nojiri & Omori, 2007
CARDO catalyzes regioselective and stereoselective dihydroxylation of the angular (C9a
) and adjacent (C1
) C atoms of carbazole. CARDO consists of a terminal oxygenase (Oxy; encoded by the carAa
gene) and the electron-transfer components ferredoxin (Fd; encoded by the carAc
gene) and ferredoxin reductase (Red; encoded by the carAd
gene). The electron required for the oxidation of carbazole is transferred from NAD(P)H to Oxy (Fig. 1). Novosphingobium
sp. KA1 possesses the carbazole-degradative plasmid pCAR3, which contains two car
gene clusters involved in carbazole assimilation and several orphan genes for electron transfer (carAaIAcI
; Urata et al.
Figure 1 Components and functions of the class IIA CARDO system. The proposed electron-transfer and dioxygenation reactions are illustrated. The cofactors of each component are shown in italics. The subscripts ‘ox’ and ‘red’ indicate (more ...)
CARDO is a member of the Rieske nonhaem iron oxygenases (ROs) that are found in various species of bacteria. ROs have been divided into five subgroups (IA, IB, IIA, IIB and III) based on the number of constituents and the nature of their redox centres (Batie et al.
). The CARDOs of KA1 are classified as class IIA ROs, while the well studied CARDOs from Pseudomonas resinovorans
sp. J3 and Nocardioides aromaticivorans
IC177 belong to classes III, III and IIB, respectively (Nojiri & Omori, 2007
). In this paper, the Oxys from KA1, CA10, J3 and IC177 are abbreviated OxyIIA
, respectively. The same method of abbreviation is used for the Fds and Reds of each strain.
Several recent studies have described the structure of RO oxygenases such as naphthalene dioxygenase (Kauppi et al.
), biphenyl dioxygenase (Furusawa et al.
), 2-oxoquinoline 8-monooxygenase (Martins et al.
) and dicamba monooxygenase (D’Ordine et al.
; Dumitru et al.
). Naphthalene dioxygenase and biphenyl dioxygenase exist as α3
heterohexamers. CARDO, 2-oxoquinoline 8-monooxygenase and dicamba monooxygenase form α3
homotrimers and phthalate dioxygenase is thought to be an α3
homohexamer (Tarasev et al.
To date, we have determined the crystal structures of several components of CARDO: FdIII
from P. resinovorans
CA10 (Nam et al.
sp. J3 (Nojiri et al.
) and the OxyIII
electron-transfer complex (Ashikawa et al.
) and OxyIIB
from N. aromaticivorans
(Inoue et al.
). We have also successfully crystallized FdIIA
sp. KA1 (Umeda et al.
) and RedIII
sp. J3 (Ashikawa et al.
) and the structures of these proteins have been solved (Ashikawa et al.
, in preparation; Umeda et al.
, in preparation).
This report describes the crystallization and preliminary X-ray diffraction study of OxyIIA (encoded by carAaII; a trimer of 383 residues with a molecular mass of 129.3 kDa).