Yuji Ashikawa

Divergent Structures of Carbazole Degradative car Operons Isolated from Gram-negative Bacteria

Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Crystal structure of the ferredoxin component of carbazole 1,9a‐dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non‐heme iron oxygenase system

The carbazole 1,9a‐dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein–protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 Å resolution by molecular replacement using the structure of BphF, the biphenyl 2,3‐dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three β‐sheets, and the structure can be divided into two domains, a cluster‐binding domain and a basal domain. The Rieske [2Fe–2S] cluster is located at the tip of the cluster‐binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster‐binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different‐featured components. Proteins 2005.

Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase

BackgroundDihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex.ResultsIn the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding.ConclusionsThe RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O–O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Specific Interactions between the Ferredoxin and Terminal Oxygenase Components of a Class IIB Rieske Nonheme Iron Oxygenase, Carbazole 1,9a-Dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (Oxy), ferredoxin (Fd), and ferredoxin reductase (Red) components and is a member of the Rieske nonheme iron oxygenases. Rieske nonheme iron oxygenases are divided into five subclasses (IA, IB, IIA, IIB, and III) based on the number of constituents and the nature of their redox centers. Each component of a class IIB CARDO from Nocardioides aromaticivorans IC177 was purified, and the interchangeability of the electron transfer reactions with each component from the class III CARDOs was investigated. Despite the fact that the Fds of both classes are Rieske-type, strict specificities between the Oxy and Fd components were observed. On the other hand, the Fd and Red components were interchangeable, even though the Red components differ in cofactor composition; the class IIB Red contains flavin-adenine-dinucleotide (FAD)- and NADH-binding domains, whereas the class III Red has a chloroplast-type [2Fe-2S] cluster in addition to the FAD- and NADH-binding domains. The crystal structures of the class IIB Oxy and Fd components were compared to the previously reported Fd:Oxy complex structure of class III CARDO. This comparison suggested residues in common between class IIB and class III CARDOs that are important for interactions between Fd and Oxy. In the class IIB CARDOs, these included His75 and Glu71 in Fd and Lys20 and Glu357 in Oxy for electrostatic interactions, and Phe74 and Pro90 in Fd and Trp21, Leu359, and Val367 in Oxy for hydrophobic interactions. The residues that formed the interacting surface but were not conserved between classes were thought to be necessary to form the appropriate geometry and to determine electron transfer specificity between Fd and Oxy.

Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 A and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 158.7, c = 81.4 A. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 A resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 A.

Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase component of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1.

Carbazole 1,9a-dioxygenase (CARDO) is the initial enzyme of the carbazole-degradation pathway. The CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase (Red) and is classified as a class IIA Rieske oxygenase. Red from KA1 was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG 4000. The crystal diffracted to 1.58 A resolution and belonged to space group P3(2), with unit-cell parameters a = b = 92.2, c = 78.6 A, alpha = gamma = 90, beta = 120 degrees . Preliminary analysis of the X-ray diffraction data revealed that the asymmetric unit contained two Red monomers. The crystal appeared to be a merohedral twin, with a twin fraction of 0.32 and twin law (-h, -k, l).

Crystallization and preliminary X-ray diffraction studies of a terminal oxygenase of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1

Carbazole 1,9a-dioxygenase (CARDO) is the initial dioxygenase in the carbazole-degradation pathway of Novosphingobium sp. KA1. The CARDO from KA1 consists of a terminal oxygenase (Oxy), a putidaredoxin-type ferredoxin and a ferredoxin reductase. The Oxy from Novosphingobium sp. KA1 was crystallized at 277 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction data were collected to a resolution of 2.1 Å. The crystals belonged to the monoclinic space group P2(1). Self-rotation function analysis suggested that the asymmetric unit contained two Oxy trimers; the Matthews coefficient and solvent content were calculated to be 5.9 Å(3) Da(-1) and 79.1%, respectively.

Crystallization and preliminary X-ray diffraction studies of a novel ferredoxin involved in the dioxygenation of carbazole by Novosphingobium sp. KA1

Novosphingobium sp. KA1 uses carbazole 1,9a-dioxygenase (CARDO) as the first dioxygenase in its carbazole-degradation pathway. The CARDO of KA1 contains a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. In contrast to the CARDO systems of other species, the ferredoxin component of KA1 is a putidaredoxin-type protein. This novel ferredoxin was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals belong to space group C222(1) and diffraction data were collected to a resolution of 1.9 A (the diffraction limit was 1.6 A).

Crystallization and preliminary X-ray diffraction studies of the terminal oxygenase component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular-position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The terminal oxygenase component (43.9 kDa) of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 8000 as the precipitant. The crystals diffract to 2.3 A resolution and belong to space group C2.