Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida.
Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.
D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D012256:Riboflavin / Q000494:pharmacology; D013053:Spectrophotometry
D000596:Amino Acids; D009097:Multienzyme Complexes; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D010105:Oxygenases; C023172:toluene dioxygenase; D012256:Riboflavin