Alteration of the Substrate Specificity of the Angular Dioxygenase Carbazole 1,9a-Dioxygenase

Uchimura, Hidemasa; Horisaki, Tadafumi; Umeda, Takashi; Noguchi, Hiroshi; Usami, Yusuke; Li, Li; Terada, Tohru; Nakamura, Shugo; Shimizu, Kentaro; Takemura, Tamiko; Habe, Hiroshi; Furihata, Kazuo; Omori, Toshio; Yamane, Hisakazu; Nojiri, Hideaki

doi:10.1271/bbb.80512

Cited by 8 publications

(9 citation statements)

References 32 publications

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“…In addition, we created CARDO-O J3 mutants having substrate specificities different from that of wild-type CARDO-O J3 . 38) The crystal structures of CARDO-O J3 mutants bound to various substrates also accord with Ferraro's hypothesis (Usami and Inoue et al, unpublished results).…”

Section: Structural Basis Of the Novel Substrate Specificity Of supporting

confidence: 71%

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

Nojiri

2012

Bioscience, Biotechnology, and Biochemistry

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Section: Structural Basis Of the Novel Substrate Specificity Of supporting

confidence: 71%

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

Nojiri

2012

Bioscience, Biotechnology, and Biochemistry

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“…To express each amino acid-substituted derivative of Oxy as a C-terminally His-tagged protein, we used the pET system with the pET-26b(ϩ) vector and Escherichia coli strain BL21(DE3) (Novagen, Madison, WI). Artificial NdeI and XhoI sites were created at the 5= and 3= ends of each carAa mutated gene, respectively, by PCR with template DNA containing the target mutant carAa gene (pUCARAJ3-I262L, -I262V, -F275W, -Q282N, or -Q282Y) (30) and primers 5=-CATATGGCGAACGTTGATGAGGCAAT-3= and 5=-CTCGA GGCCCGAAACGTGCGCTTGGGTCTGAATACCCTG-3= (the NdeI and XhoI restriction sites are italicized). The nucleotide sequences of the resultant PCR amplicons were confirmed by sequencing analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies of CARDO demonstrated that site-directed mutagenesis of the hydrophobic amino acids located at the surface of the substratebinding pocket alters the substrate specificity of the enzyme (30). In the crystal structure of WT-CAR, amino acid residues Leu202 to Thr214 and Asp229 to Val238 near the entrance of the substrate-binding pocket are shifted inward (26).…”

Section: Structures Of Oxy With Amino Acid Replacement At Gln282mentioning

confidence: 99%

“…The product formation rates of the biotransformation study previously performed with WT CARDO and its amino acid-substituted derivatives generated by site-directed mutagenesis (30) are indicated on the right. The product formation rates were calculated from the peak areas for the total ion current of the respective compounds extracted from the reaction mixtures containing E. coli cells expressing the WT or derivative CARDOs by gas chromatography-mass spectrometry analyses (30). Percentages of angular and lateral dioxygenation products are in parentheses.…”

Section: Figmentioning

confidence: 99%

“…To investigate the structural properties that determine the substrate specificity of CARDO, amino acid-substituted derivatives of Oxy have been generated by site-directed mutagenesis and their specificities for hetero-and polyaromatic substrates were determined (30). Four amino acid residues, Ile262, Phe275, Gln282, and Phe329, were selected for mutation on the basis of simulations of Oxy docking with substrates.…”

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Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

Inoue

Usami

Ashikawa

et al. 2014

Appl Environ Microbiol

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e Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substratebinding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates. Rieske nonheme iron oxygenases (ROs) catalyze the initial oxygenation reaction of aromatic compounds (1). ROs are of interest for the biodegradation of aromatic pollutants and for synthetic applications requiring enantio-and regiospecific reactions. Some ROs break down toxic heterocyclic compounds by a single oxidization reaction. ROs are generally composed of two or three components, a terminal oxygenase (Oxy) and one or two electron transfer components. The Oxy is activated by the electron transfer component(s) and then catalyzes the oxygenation of the substrate. Recent biochemical and structural studies have revealed that Oxys typically exhibit an ␣ 3 or (␣␤) 3 configuration (2). The ␣ subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron. The Rieske [2Fe-2S] cluster accepts electrons from the electron transfer component(s) and transfers electrons to the mononuclear iron. The mononuclear iron at the active site activates molecular oxygen, which attacks the substrate. The two oxygen atoms of the molecular oxygen bind to tandemly linked carbon atoms in an aromatic ring, creating two hydroxyl groups in the cis configuration (3). This process is called lateral dioxygenation. The amino acid residues that affect the substrate specificity of ROs have bee...

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carbazole 1,9a-dioxygenase 1.14.12.22

Schomburg¹,

Schomburg²

2013

Class 1 Oxidoreductases

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Alteration of the Substrate Specificity of the Angular Dioxygenase Carbazole 1,9a-Dioxygenase

Cited by 8 publications

References 32 publications

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

carbazole 1,9a-dioxygenase 1.14.12.22

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