Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2.
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.
D000645:Ammonium Sulfate; D002384:Catalysis; D002614:Chelating Agents; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003065:Coenzyme A; D003300:Copper; D004586:Electrophoresis; D005663:Furans; D007501:Iron / Q000032:analysis; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D008970:Molecular Weight; D010100:Oxygen; D011134:Polysaccharides; D011549:Pseudomonas / Q000201:enzymology*; D014867:Water
D002614:Chelating Agents; D005663:Furans; D011134:Polysaccharides; D014867:Water; D003300:Copper; D007501:Iron; D006899:Mixed Function Oxygenases; D010100:Oxygen; D003065:Coenzyme A; D000645:Ammonium Sulfate