Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1

Abstract

The toluene/o-xylene monooxygenase cloned from Pseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been 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 similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase in Escherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/ o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

FIG. 1

Reactions (A) catalyzed by and genetic map (B and C) of the P. stutzeri OX1 locus coding for toluene/o-xylene monooxygenase. (A) I, R=H, toluene; R=CH₃, o-xylene; II, R=H, o-, m-, or p-cresol; R=CH₃, 2,3- or 3,4-DMP; III, R=H, 3-methylcatechol (4-methylcatechol can also be formed from cresols); R=CH₃, 3,4-dimethylcatechol. (B) Restriction endonuclease map of the DNA fragment expressing toluene/o-xylene monooxygenase activity (pBZ1260). Striped boxes represent the vector polylinker with relevant cloning sites. Sl, SalI; K, KpnI; Av, AvaI; N, NotI; Sm, SmaI; D, DraI; Bm, BamHI; Xb, XbaI; S, SalI. (C) The boxes indicate the ORFs identified (also see Table 2), and the points of the arrows indicate the direction of the transcription. In panel D, the mutations introduced in each ORF in turn are schematized (further details are reported in Materials and Methods).