Diverse oxygenations catalyzed by carbazole 1,9a-dioxygenase from Pseudomonas sp. Strain CA10

Abstract

Carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-p-dioxin. It was revealed by gas chromatography-mass spectrometry and 1H and 13C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2',3-trihydroxydiphenylmethane and 2,2',3-trihydroxydiphenyl sulfide, respectively. Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds. In addition to the angular dioxygenation, CARDO catalyzed the cis dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl. Naphthalene and biphenyl were converted by CARDO to cis-1, 2-dihydroxy-1,2-dihydronaphthalene and cis-2,3-dihydroxy-2, 3-dihydrobiphenyl, respectively. On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively. These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation, cis dihydroxylation, and monooxygenation. The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO.

FIG. 1

Conversion of CAR (A), DD (B), and DBF (C) catalyzed by CARDO from Pseudomonas sp. strain CA10. The structures shown in brackets are unstable intermediates that have not been characterized. Absolute stereochemistry is not intended.

FIG. 2

Oxidation reaction catalyzed by CARDO from Pseudomonas sp. strain CA10 and identified by GC-MS or NMR analysis for xanthene (A), phenoxathiin (B), dibenzothiophene (C), naphthalene (D), anthracene (E), fluorene (F), fluoranthene (G), and biphenyl (H). The compounds shown in brackets are the hypothetical products generated by CARDO. Monohydroxylated compounds were also identified in the biotransformation experiments with dibenzothiophene, naphthalene, anthracene, fluorene, fluoranthene, and biphenyl. The presence of cis-7,8-dihydroxy-7,8-dihydrofluoranthene and fluorene dihydrodiols was presumed because of the identification of 7-hydroxyfluoranthene and monohydroxyfluorenes. Absolute stereochemistry is not intended.

FIG. 3

Proposed initial attack on CAR (A) and the CAR analogues DD (B), dibenzothiophene (C), and fluorene (D) by CARDO from Pseudomonas sp. strain CA10. The small arrows indicate the positions of the enzymatic incorporation of oxygen. The structures shown in brackets are unstable intermediates that have not been characterized. Absolute stereochemistry is not intended. The shaded areas represent the structure of anthracene or DD.

FIG. 4

Proposed initial attack on fluoranthene (A), naphthalene (B), and biphenyl (C) by CARDO from Pseudomonas sp. strain CA10. The small arrows indicate the positions of the enzymatic incorporation of oxygen. The structures shown in brackets are the hypothetical products of cis hydroxylation by CARDO that have not been characterized. Absolute stereochemistry is not intended. The shaded areas represent the structure of anthracene or DD.