BIODEGRADATION OF 4-METHYL-5-NITROCATECHOL BY PSEUDOMONAS SP STRAIN DNT

Pseudomonas sp. strain DNT degrades 2,4-dinitrotoluene (DNT) by a diox ygenase attack at the 4,5 position with concomitant removal of the nit ro group to yield 4-methyl-5-nitrocatechol (MNC). Here we describe the mechanism of removal of the nitro group from MNC and subsequent react ions leading to ring fission. Washed suspensions of DNT-grown cells ox idized MNC and 2,4,5-trihydroxytoluene (THT). Extracts prepared from D NT-induced cells catalyzed the disappearance of MNC in the presence of oxygen and NADPH. Partially purified MNC oxygenase oxidized MNC in a reaction requiring 1 mol of NADPH and 1 mol of oxygen per mol of subst rate. The enzyme converted MNC to 2-hydroxy-5-methylquinone (HMQ), whi ch was identified by gas chromatography-mass spectrometry. HMQ was als o detected transiently in culture fluids of cells grown on DNT. A quin one reductase was partially purified and shown to convert HMQ to THT i n a reaction requiring NADH. A partially purified THT oxygenase cataly zed ring fission of THT and accumulation of a compound tentatively ide ntified as 3-hydroxy-5-(1-formylethylidene)-2-furanone. Preliminary re sults indicate that this compound is an artifact of the isolation proc edure and suggest that 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic ac id is the actual ring fission product.