Alteration of the substrate specificity of the angular dioxygenase carbazole 1,9a-dioxygenase.
Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.
Uchimura
Hiromasa
H
Biotechnology Research Center, The University of Tokyo, Tokyo, Japan.
Horisaki
Tadafumi
T
Umeda
Takashi
T
Noguchi
Haruko
H
Usami
Yusuke
Y
Li
Li
L
Terada
Tohru
T
Nakamura
Shugo
S
Shimizu
Kentaro
K
Takemura
Tetsuo
T
Habe
Hiroshi
H
Furihata
Kazuo
K
Omori
Toshio
T
Yamane
Hisakazu
H
Nojiri
Hideaki
H
D000873:Anthracenes / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002227:Carbazoles / Q000378:metabolism; D020134:Catalytic Domain; D004147:Dioxins / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005449:Fluorenes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013379:Substrate Specificity; D013876:Thiophenes / Q000378:metabolism
D000873:Anthracenes; D001426:Bacterial Proteins; D002227:Carbazoles; D004147:Dioxins; D005449:Fluorenes; D013876:Thiophenes; C041514:carbazole; C007738:fluoranthene; C041509:fluorene; D049308:Dioxygenases; C108434:carbazole 1,9a-dioxygenase; C034020:anthracene; C020887:dibenzo(1,4)dioxin; C016366:dibenzothiophene