4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2

Abstract

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases

“…Protein was routinely determined by the method of Bradford (Bradford 1976), with bovine serum albumin as standard. The molecular mass of native PmdAB was assayed by gel-filtration chromatography on a Superose 12 column (Pharmacia) with the mobile phase described in step 2 of the gel filtration chromatography procedure (see above) at a flow rate of 0.4 ml/min; standard proteins are described elsewhere (Locher et al 1991). SDS-PAGE was done with 12% separative gels (Scha¨gger and von Jagow 1987), and proteins were stained with colloidal Coomassie Brilliant Blue G-250 (Neuhoff et al 1988); a 10-kDa Protein Ladder (Gibco) was used for calibration.…”

“…The activity of PmdAB was assayed routinely at 30°C as substrate-dependent oxygen uptake in a Clark-type oxygen electrode (Locher et al 1991). Reaction mixtures (0.5 ml) contained 40 mM Tris-SO 4 (pH 7.5), 0.5-1.5 mg protein and 2.5 lmol PCA, with which the reaction was started.…”

Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system

“…were from commercial sources; THB was prepared by H. Harms (9). The sources of other chemicals are reported elsewhere (9,17,18). DEAE-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) was used in addition to the protein liquid chromatography columns described previously (17,18).…”

“…THB was sometimes silylated and examined in a mass spectrometer (model ITD 800; Finnigan, San Jose, Calif.) coupled to a gas chromatograph (model HRGC 5160; Carlo Erba, Rodano, Italy); compounds were separated on a 10-m PS 090 glass capillary column. Iron, by atomic absorption spectroscopy, and inorganic sulfur, as methylene blue, were determined as described elsewhere (17). N-terminal amino acid sequences were determined as detailed by Locher et al (18 …”

Microbial metabolism of sulfurand phosphorus-containing xenobiotics

“…7a). An enzyme of this type, catalysing degradation of p-sulfobenzoic acid to protocatechuic acid in Comamonas testosteroni, has been isolated and characterized [85], and the same mechanism has been found for desulfonation of benzenesulfonate and toluenesulfonate in an Alcaligenes sp. [86,87], and for naphthalenesulfonate metabolism in Pseudomonas and Moraxella species [88][89][90].…”