Toluene monooxygenase from the fungus Cladosporium sphaerospermum

Assimilation of toluene by Cladosporium sphaerospermum is initially catalyzed by toluene monooxygenase (TOMO). TOMO activity was induced by adding toluene to a glucose-pregrown culture of C. sphaerospermum. The corresponding microsomal enzyme needed NADPH and O(2) to oxidize toluene and glycerol, EDTA, DTT, and PMSF for stabilization. TOMO activity was maximal at 35 degrees C and pH 7.5 and was inhibited by carbon monoxide, Metyrapone, and cytochrome c. TOMO preferred as substrates also other aromatic hydrocarbons with a short aliphatic side chain. Its reduced carbon monoxide difference spectrum showed a maximum at 451 nm. A substrate-induced Type I spectrum was observed on addition of toluene. These results indicated that TOMO is a cytochrome P450. TOMO and its corresponding reductase were eventually purified by a simultaneous purification revealing apparent molecular masses of 58 and 78 kDa, respectively.