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 °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.