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

Winfried A Suske,Martin Held,Andreas Schmid,Thomas Fleischmann,Marcel G Wubbolts,Hans-Peter E Kohler

doi:10.1074/jbc.272.39.24257

Winfried A Suske, Martin Held + Show 4 more

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https://doi.org/10.1074/jbc.272.39.24257

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Abstract

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 Km values (30 degrees C, pH 7.5) were 2.8 microM for 2-hydroxybiphenyl, 26.8 microM for NADH, and 29.2 microM 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 betaalphabeta-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.

Highlights

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 microbial degradation of 2-hydroxy- and 2,2Јdihydroxybiphenyl has become important to researchers involved in the desulfurization of coal and petroleum, since it was reported that 2-hydroxy- and 2,2Ј-dihydroxybiphenyl are the end products of the bacterial desulfurization of dibenzothiophene, a major sulfur-containing component of fossil fuels (14 – 16)
We report on the purification and characterization of the enzyme. 2-Hydroxybiphenyl 3-monooxygenase is a novel flavin-containing, NADH-dependent aromatic hydroxylase with a broad substrate spectrum

Summary

MATERIALS AND METHODS

P. azelaica HBP1 was cultured on 2-hydroxybiphenyl as described previously [2]. Cultures were grown in Erlenmeyer flasks on a rotary shaker (150 –170 rpm) at 30 °C. First Anion Exchange Chromatography—The supernatant of the above purification step was directly loaded onto an anion exchange column (1 ϫ 15 cm; Fractogel EMD TMAE-650; Merck, Darmstadt, Germany) equilibrated with 10 mM triethanolamine-HCl buffer (pH 7.5). Second Anion Exchange Chromatography—The desalted enzyme solution was supplemented with FAD to a final concentration of 0.3 mM and loaded onto an anion exchange column (Fractogel EMD TMAE-650) equilibrated with 10 mM triethanolamine-HCl buffer (pH 8.2). Gel Filtration—The fractions containing active enzyme from the preceding step were incubated with 0.3 mM FAD for 30 min on ice. The solution was passed with a flow rate of 1.5 ml minϪ1 through a Superdex 200 gel filtration column (1.6 ϫ 60 cm; Pharmacia) equilibrated with 50 mM Na2HPO4 buffer (pH 7.5). The investigation of the pH dependence was done with the following buffers of constant ionic strength (I ϭ 20 mM): 20 mM MES,, pH 6 – 6.75; 20 mM Na2HPO4/KH2PO4, pH 6.75–7.75; 20 mM Tris-HCl, pH 7.75– 8.5

Analytical Methods

RESULTS

DISCUSSION