OXIDATIVE RELEASE OF NITRITE FROM 2-NITROTOLUENE BY A 3-COMPONENT ENZYME-SYSTEM FROM PSEUDOMONAS SP STRAIN-JS']JS42

Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase(2NT)) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin(NAP) from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin(2NT). Experiments with O-18(2) showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.