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Volume 272, Number 39, Issue of September 26, 1997 pp. 24257-24265
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Characterization of 2-Hydroxybiphenyl 3-Monooxygenase, a Novel NADH-dependent, FAD-containing Aromatic Hydroxylase from Pseudomonas azelaica HBP1

(Received for publication, January 31, 1997, and in revised form, July 19, 1997)

Winfried A. Suske , Martin Held ^§ , Andreas Schmid ^¶ , Thomas Fleischmann , Marcel G. Wubbolts ^§ and Hans-Peter E. Kohler

From the  Department of Microbiology, Swiss Federal Institute of Environmental Sciences and Technology, CH-8600 Dübendorf, Switzerland, the ^§ Institute of Biotechnology, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland, and the ^¶ Department of Biological Waste Air Purification, University of Stuttgart, D-70569 Stuttgart, Germany

2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1, 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 coli JM109, which efficiently expressed the hbpA gene. 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 apparent K_m values (30 °C, pH 7.5) were 2.8 µM for 2-hydroxybiphenyl, 26.8 µM for NADH, and 29.2 µM for oxygen. The enzyme was inactivated by p-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 -fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.

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