CHARACTERIZATION OF 2,2',3-TRIHYDROXYBIPHENYL DIOXYGENASE, AN EXTRADIOL DIOXYGENASE FROM THE DIBENZOFURAN-P-DIOXIN-DEGRADING AND DIBENZO-P-DIOXIN-DEGRADING BACTERIUM SPHINGOMONAS SP STRAIN RW1

A key enzyme in the degradation pathways of dibenzo-p-dioxin and diben zofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is respo nsible for meta cleavage of the first aromatic ring, has been genetica lly 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. Wi lkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microb iol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of thi s enzyme is typical of those of extradiol dioxygenases. This enzyme, w hich is extremely oxygen labile, was purified anaerobically to apparen t 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 diox ygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar K(m) values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong s ubstrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-meth ylcatechol, and 4-methylcatechol were oxidized less efficiently and 3, 4-dihydroxybiphenyl was oxidized considerably less efficiently.