Purification and properties of protocatechuate 3,4-dioxygenase from Pseudomonas putida. A new iron to subunit stoichiometry.
Protocatechuate dioxygenase has been isolated from Pseudomonas putida. This new species of protocatechuate dioxygenase has been characterized and compared with the enzyme from Pseudomonas aeruginosa. The enzyme reported here has visible absorption, circular dichroism, electron paramagnetic resonance, and Raman spectroscopic properties virtually identical to those for protocatechuate dioxygenase from P. aeruginosa. However, the molecular weight and iron:subunit stoichiometry differ. Protocatechuate dioxygenase from P. putida has a molecular weight of 200,000 and contains 4 alpha subunits of 23,000 daltons, 4 beta subunits of 26,500 daltons, and 4 ferric irons suggesting that the enzyme is a tetramer of (alpha beta Fe+3) catalytic units. Protocatechuate dioxygenase from P. aeruginosa has been reported to have a Mr = 700,000, consisting of 16 alpha subunits of 22,500 daltons, 16 beta subunits of 25,000 daltons, and 8 ferric irons (Yoshida, R., Hori, K., Fujiwara, M., Saeki, Y., Kagamiyama, H., and Nozaki, W. (1976) Biochemistry 15, 4048-4053). This enzyme is thought to be an octamer of (alpha 2 beta 2 Fe+3) catalytic units, although reconstitution with extra iron will somewhat increase its activity. Using stopped flow techniques, we have shown that essentially all of the iron in the P. putida enzyme is catalytically active. This suggests that the minimal catalytic unit of all non-heme iron intradiol dioxygenases is an (alpha beta Fe+3) structure.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification*; D011487:Protein Conformation; D011520:Protocatechuate-3,4-Dioxygenase / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D000596:Amino Acids; D046911:Macromolecular Substances; D007501:Iron; D010105:Oxygenases; D011520:Protocatechuate-3,4-Dioxygenase