Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1.
A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.
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Department of Microbiology, GBF-National Research Center for Biotechnology, Braunschweig, Germany.
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Hedderich
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Timmis
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D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001572:Benzofurans / Q000378:metabolism*; D003360:Cosmids; D004269:DNA, Bacterial / Q000302:isolation & purification; D004147:Dioxins / Q000378:metabolism*; D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001572:Benzofurans; D004269:DNA, Bacterial; D004147:Dioxins; C023614:dibenzofuran; D010105:Oxygenases; C084169:2,2',3-trihydroxybiphenyl dioxygenase; D049308:Dioxygenases; C020887:dibenzo(1,4)dioxin