Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2

Abstract

1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH(4))(2)SO(4) fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Delta(2)-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27x10(6) and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5-9.5, a K(m) for 2-furoyl-CoA of 20.2mum and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.

Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition

“…The presence of saccharose (10%) in the buffers stabilized XDH activity to some extent when the antibody column was used. Addition of 100 mM ammonium sulfate and 1 mM iodoacetamide (23) Determination of purity and molecular mass. Electrophoresis of the purified larger and smaller 185W-labeled proteins on SDS-polyacrylamide gels resulted in both cases in two protein bands with molecular weights of 55,000 and 25,000 (Fig.…”

“…Aerobic degradation of 2-furoic acid involves the formation of 2-furoyl-CoA followed by hydroxylation to form 5-hydroxy-2-furoyl-CoA (23,24,50). The hydroxyl group derives from water rather than from molecular oxygen (23).…”

“…Molybdoenzymes oxidized by KI and I2 showed the typical blue fluorescence of the oxidized pterin as part of the molybdenum cofactor (23). XDH or FCoADH solution (500 ,ul; 0.5 mg/ml) was adjusted to pH 2.5 with 15% phosphoric acid.…”

“…Unlike the 2-furoyl-CoA dehydrogenase (FCoADH), the xanthine dehydrogenase (XDH) is a constitutive, separate enzyme that also can carry out an FCoADH reaction (24). Although a FCoADH from a different strain of P. putida was described as containing supposedly copper but no iron or flavin (23), the reactions catalyzed by this enzyme seem to be quite analogous to those catalyzed by molybdenum-containing hydroxylases (2,9,15). In all of these cases, the hydroxyl group derives from water (2,23; W. C. Evans, Biochem.…”

“…The formation of 2-furoyl-coenzyme A (CoA) is the first reaction step in 2-furoic acid degradation. The second enzymatic step is catalyzed by a dehydrogenase that introduces a hydroxyl group to form 5-hydroxy-2-furoylCoA (23,24,49,50). Other substrates for P. putida Ful were furfuryl alcohol and furfural, which were degraded via 2-furoic acid and 2-furoyl-CoA (Fig.…”