PURIFICATION AND PROPERTIES OF NADH-FERREDOXINTOL REDUCTASE - A COMPONENT OF TOLUENE DIOXYGENASE FROM PSEUDOMONAS-PUTIDA

  • 1 January 1981
    • journal article
    • research article
    • Vol. 256  (6) , 2723-2730
Abstract
Cells of P. putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methylcyclohexa-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. Equilibrium dialysis experiments showed that the enzyme can bind 1 mol of FAD/mol of enzyme protein. The apparent MW 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.