Purification and Characterization of Carbazole 1,9a-Dioxygenase, a Three-Component Dioxygenase System of Pseudomonas resinovorans Strain CA10

Abstract

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.Pseudomonas resinovorans strain CA10 is a bacterium that has the ability to utilize carbazole (CAR) as its sole source of carbon, nitrogen, and energy. As the initial degradation reaction, CAR is dioxygenated at the angular (C-9a) and adjacent (C-1) positions to yield the unstable cis-hydrodiol (25, 27). The resultant cis-hydrodiol is spontaneously converted to 2Ј-aminobiphenyl-2,3-diol, which is further converted to anthranilate via meta cleavage and hydrolysis. Such initial dioxygenation is called angular dioxygenation and contains the degradation pathways of CAR and its structural analogues (25).Genes encoding CAR 1,9a-dioxygenase (CARDO) were cloned from P. resinovorans strain CA10, and CARDO was found to be a multicomponent enzyme system which consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd) (32). Amino acid sequence homology and phylogenetic analysis revealed that CarAa is a rather unique type of oxygenase that shares low homology with other known dioxygenases (23, 32). Furthermore, from studies on substrate specificity, it was found that CARDO has a broad substrate range and can catalyze diverse oxygenations: angular dioxygenation, lateral dioxygenation, and monooxygenation (26). In addition, Habe et al. (11) have reported that CARDO can attack some chlorinated dioxin congeners in a manner similar to that of dibenzofuran 4,4a-dioxygenase from Terrabacter sp. strain DBF63 and dioxin dioxygenase from Sphingomonas wittichii strain RW1 (43), although some difference was observed in particular features. Compared to other degradative reactions for dio...

Isolation and Description of a Stable Carbazole-Degrading Microbial Consortium Consisting of Chryseobacterium sp. NCY and Achromobacter sp. NCW

“…This was in agreement with the previous report by Kohtaro, et al [7]. The initial catalyzing reaction involved in the biodegradation of carbazole was carried by the CARDO system, which contained three components: terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd) [11]. The gene encoding CARDO was located on a 199-kb circular megaplasmid pCAR1 [2].…”

Aerobic Degradation of Petroleum Components by Microbial Consortia

“…The initial reactions of dibenzofuran and carbazole can be classified into angular and lateral dioxygenation, which may be catalyzed by different enzymes. These enzymes were found in Gram-positive and negative bacteria [138,152,[154][155][156]. Many bacterial strains have the ability to use carbazole as the sole carbon, nitrogen and energy source [150,157].…”

“…Heteroaromatic compounds: Many bacterial species have been reported to decompose dibenzofurans and carbazole, a structural analogue with nitrogen instead of oxygen [149][150][151][152][153]. Guo et al [151] isolated a stable carbazole-degrading microbial consortium consisting of Chryseobacterium sp.…”

Properties, environmental fate and biodegradation of carbazole

“…The extensively studied CARDO system in Pseudomonas resinovorans CA10 is a three-component dioxygenase system belonging to the Rieske nonheme iron oxygenase system (ROS) and consist of a terminal oxygenase and electron transport proteins (Sato et al 1997a;Nam et al 2002). The terminal oxygenase component of CARDO (CARDO-O) is a homotrimeric enzyme that contains one Rieske [2Fe-2S] cluster ([2Fe-2S] R and one active-site iron (Fe 2? )…”

“…in a single subunit (CarAa) (Nojiri and Omori 2007). The electron transport proteins of CARDO, which mediate electron transport from NAD(P)H to CARDO-O, comprise ferredoxin (CARDO-F; a monomer of CarAc), which contains one [2Fe-2S] R , and ferredoxin reductase (CARDO-R; a monomer of CarAd), which contains one FAD and one plant-type [2Fe-2S] cluster ([2Fe-2S] P ) (Sato et al 1997a;Nam et al 2002).…”