| Abstrakt: |
2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation inPseudomonas azelaicaHBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl. The enzyme was also purified from a recombinant of Escherichia coliJM109, which efficiently expressed the hbpAgene. Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations. Gel filtration, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular mass of 256 kDa. Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD. The monooxygenase had a pI of 6.3. It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. Molecular oxygen was the source of the additional oxygen of the product. The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group. All substrates effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide. 2,3-Dihydroxybiphenyl, the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation and hydrogen peroxide formation without being hydroxylated and also was an inhibitor. The apparentKmvalues (30 °C, pH 7.5) were 2.8 μmfor 2-hydroxybiphenyl, 26.8 μmfor NADH, and 29.2 μmfor oxygen. The enzyme was inactivated byp-hydroxymercuribenzoate, a cysteine-blocking reagent. In the presence of 2-hydroxybiphenyl, the enzyme was partly protected against the inactivation, which was reversed by the addition of an excess of dithiothreitol. The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the βαβ-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonasstrain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha. |
