The nucleotide sequence of the locus coding for P. stutzeri OX1 toluene/o-xylene monooxygenase revealed six ORFs, designated touABCDEF, which showed relevant similarities to the subunits of several enzymatic complexes involved in the monooxygenation of aromatic compounds. Each gene found in the locus was shown to be essential for the full enzymatic activity. These results provide genetic evidence that toluene/o-xylene monooxygenase, the enzyme responsible for the initial steps of toluene and o-xylene catabolism in P. stutzeri OX1, is a multicomponent monooxygenase.
The gene cluster encoding the P. stutzeri
-xylene monooxygenase has a GC ratio similar to, and the same gene arrangement as, the tbu
, and bmo
operons of the toluene monooxygenases from B. pickettii
), P. mendocina
), B. cepacia
), and P. aeruginosa
). These data, together with the presence of a putative transposase (ORF A), suggest that these genes might have been recently acquired by gene transfer from other bacteria. Further investigations are required to confirm this hypothesis.
Comparison of Tou polypeptides with those belonging to more-characterized systems led us to hypothesize for them a role in a four-component monooxygenase.
TouF and TouC may represent the components of the electron transport chain. TouF is presumably necessary for NADH oxidation and for the transport of the two reduction equivalents to the central Rieske-type ferredoxin (TouC). ORFs having the Rieske-type motif or ferredoxin-like motifs were found in virtually all of the aromatic compound-hydroxylating complexes. In the two-component systems, the NADH-ferredoxin reductase activity is due to a single component (i.e., XylA or BenC) that probably evolved from the fusion of an NADH reductase with a ferredoxin (27
). In three- or four-component systems, a Rieske-type ferredoxin that transfers the electrons from the NADH reductase to the terminal oxygenase is present. The Rieske-type ferredoxin was found to be essential for reconstruction of NADH-dependent catalytic activity of T4MO in vitro, by mediating electron transfer between the reductase and the hydroxylase (33
). In the cloned P. stutzeri
OX1 monooxygenase, the knockout of either touF
did not lead to a complete loss of activity. Due to their role, it may be suggested that both functions can be at least partially accomplished by host proteins.
For polypeptides such as TouD, a regulatory function of the catalysis has been postulated (3
) but has not yet been demonstrated. In the case of DmpM protein from Pseudomonas putida
CF600, its interaction with both the hydroxylase component and phenol has been suggested (35
). Pikus et al. (33
) demonstrated that TmoD is a high-affinity component of the T4MO complex rather than a subunit of the hydroxylase and suggested that it may have a role related to catalysis. Consistent with these hypotheses, in our in vivo experiments, the knockout of the touD
gene led to a complete loss of activity, and complementation with the wild-type touD
made it possible to rescue 60 to 100% of the wild-type activity.
TouA, TouB, and TouE may represent the three peptides constituting the catalytic subunit of the enzymatic complex. Indeed, the knockout of each of the corresponding genes led to a complete loss of activity with every substrate. A three-polypeptide terminal oxygenase was also found in the P. putida
CF600 phenol hydroxylase (34
) and the B. cepacia
G4 T2MO complex (28
), but TouB and similar peptides seem to characterize the monooxygenases active on toluene and benzene.
Further proof that toluene/o
-xylene monooxygenase from P. stutzeri
OX1 is closely related to toluene monooxygenases comes from phylogenetic analysis (not shown) of the large and small subunits of the terminal hydroxylase component of several multicomponent monooxygenases. In fact, the TouA and TouE proteins from P. stutzeri
OX1 are included in the same group as BMO, T3MO, and T4MO from P. aeruginosa
), B. pickettii
), B. cepacia
), and P. mendocina
), which could be defined as a toluene subfamily. Especially in the case of the large subunit, similar subgroups can be recognized for phenol and methane monooxygenases. The only toluene monooxygenase which appears to be an exception is Tb2MO from Pseudomonas
sp. strain JS150, which, despite its activity on hydrocarbons, was previously found to be more similar to phenol monooxygenases than to toluene monooxygenases (18
Based on the genetic analysis, the P. stutzeri
-xylene monooxygenase can be considered to belong to a toluene monooxygenase subfamily; however, it is peculiar from a biochemical point of view. In fact, in comparison to the other toluene monooxygenases, it displays a broader range of substrates, recognizing both hydrocarbons and phenols, and a more relaxed regioselectivity of aromatic ring hydroxylation, being able to hydroxylate more than one position on both natural and nonnatural substrates (2
). It thus combines the specificity and the regioselectivity of all of the enzymes belonging to the toluene monooxygenase subfamily and of Tb2MO from Pseudomonas
sp. strain JS150 (18
), T2MO from B. cepacia
), and multicomponent phenol hydroxylases (9
Despite their genetic similarity, the enzymatic systems belonging to the toluene monooxygenase subfamily are thus shown to display different regioselectivities and different substrate specificities. Further efforts are under way to isolate determinants that affect their biochemical properties.