ANALYSIS OF THE GENE-CLUSTER ENCODING TOLUENE O-XYLENE MONOOXYGENASE FROM PSEUDOMONAS-STUTZERI OX1/

The toluene/o-xylene monooxygenase cloned from Pseudomonas stutzeri OX 1 displays a very broad range of substrates and a very peculiar regios electivity, because it is able to hydroxylate more than one position o n the aromatic ring of several hydrocarbons and phenols. The nucleotid e sequence of the gene cluster coding for this enzymatic system has be en determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene -degradative pathways cloned from Pseudomonas strains. Significant sim ilarities were also found with multicomponent monooxygenase systems fo r phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with t he mild type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase in Escherichia col i. This analysis also shows that despite its activity on both hydrocar bons and phenols, toluene/o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenas es active on phenols.