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.