We have recently found that toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1 (encoded by tbuA1UBVA2C) performs successive hydroxylations of benzene (Appl. Environ. Microbiol. 70: 3814, 2004) as well as hydroxylates toluene to a mixture of 90% p-cresol and 10% m -cresol which are then further oxidized to 100% 4-methylcatechol (J. Bacteriol. 186: 3117, 2004) whereas it was thought previously that TpMO forms 100% m-cresol and is not capable of successive hydroxylations. Here we propose a modification of the degradation pathway originally described by Olsen et al. (J. Bacteriol. 176: 3749, 1994) that now relies primarily on TpMO for conversion of toluene to 4-methylcatechol (instead of m-cresol) since both m-cresol and p-cresol are shown here to be good substrates for Escherichia coli expressing TpMO (Vmax/Km=0.046,0.036, and 0.055 mL min-1 mg-1 protein for the oxidation of toluene, m-cresol, and p-cresol, respectively). In light of the broader activity of TpMO, phenol hydroxylase (encoded by tbuD) appears to facilitate conversion of any m-cresol or p-cresol formed from toluene oxidation by TpMO to 4-methylcatechol; hence, the cell has a redundant method for making this important intermediate 4-methylcatechol. Further, it is suggested that the physiological relevance of the 10% m-cresol formed from toluene oxidation by TpMO is needed for induction of the meta cleavage operon tbuWEFGKIHJ to enable full metabolism of toluene since p-cresol (and o-cresol) do not induce the meta -cleavage pathway. Therefore both the successive hydroxylation of toluene by TpMO and the product distribution are of physiological relevance to the cell.
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