Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400

Abstract

The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISP BPH ) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (␣) subunit with an estimated molecular weight of 53,300 and a small (␤) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an ␣ 3 ␤ 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of ␣␤ heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxin BPH and ferredoxin BPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at g x ‫؍‬ 1.74, g y ‫؍‬ 1.92, and g z ‫؍‬ 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISP BPH in the presence of ferredoxin BPH , ferredoxin BPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISP BPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.

Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400

“…The enzyme was isolated from biphenyl-grown cells of LB400 and resolved into three components (A, B, and C) by ion-exchange chromatography as previously described (21). A preliminary account of the purification of the terminal oxygenase (component C, ISP BPH ) to homogeneity has been given (20), and the details will be published elsewhere. Component B (ferredoxin BPH ) was further purified by gel filtration chromatography on a column (1 by 30 cm) of Superose 12.…”

Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

“…Biphenyl dioxygenase (BPDO) 1 catalyzes the first reaction of the biphenyl catabolic pathway. BPDO comprises three components (1, 2): The iron-sulfur oxygenase (ISP BPH ) made up of an ␣ subunit (M r ϭ 51,000) and a ␤ subunit (M r ϭ 22,000), the ferredoxin (FER BPH , M r ϭ 12,000), and the ferredoxin reductase (RED BPH , M r ϭ 43,000).…”

Construction of Overexpression Vectors and Purification of the Oxygenase Component of Alkylphenol Hydroxylase of Pseudomonas alkylphenolia

“…No other distinctive contamination bands were observed. The native molecular weight of the protein was estimated to be approximately 220 -250 kDa from gel permeation chromatography when determined by the method previously described (Haddock and Gibson, 1995). Thus, oxygenase appears to exist as a dimer form, consistent to the crystal structures of BMM PH and toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1 (Sazinsky et al, 2004.…”

“…A shoulder was observed at 291 nm, a characteristic of these proteins, indicating a high purity since contamination of other proteins would shield the shoulder absorbance. The estimated iron content was 1.85 mole per αβγ mole as determined by the method previously described (Haddock and Gibson, 1995) and addition of ferrous iron in the assay did not stimulate the activity, indicating almost a stoichiometric amount of iron was bound as a diiron cluster at the active sites of the purified oxygenase. The purified active oxygenase is currently exploited for crystal formation to characterize aPH BMM with unique substrate selectivity and regiospecificity.…”