Regiospecificity of two multicomponent monooxygenases from Pseudomonas stutzeri OX1: molecular basis for catabolic adaptation of this microorganism to methylated aromatic compounds

Abstract

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.

FIG. 1.

Proposed pathways for the conversion of toluene to methylcatechols (A) and for the conversion of o-xylene to dimethylphenols (B) catalyzed by ToMO and PH mixtures. The thickness of the arrows is roughly proportional to the relative abundance of each species. o-C, o-cresol; m-C, m-cresol; p-C, p-cresol.

FIG. 2.

Kinetics of production of o-cresol (□), m- and p-cresols (▪), 3-methylcatechol (○), and 4-methylcatechol (•) by cells expressing ToMO at a concentration of 1 mU/ml (A), cells expressing PH at a concentration of 1 mU/ml (B), and a mixture of cells expressing ToMO (1 mU/ml) and cells expressing PH (1 mU/ml) (C) incubated with 30 μM toluene.

FIG. 3.

Rates of formation of 3-MC (•) and 4-MC (○) by mixtures of cells expressing PH (0.29 mU/ml) and cells expressing ToMO (0.29 to 2.3 mU/ml) incubated with 30 μM toluene. The concentrations of 3-MC and 4-MC were measured by HPLC as described in Materials and Methods.

FIG. 4.

Kinetics of production of 2,3-DMP (□), 3,4-DMP (▪), 3,4-DMC (○), and 4,5-DMC (•) by cells expressing ToMO at a concentration of 1.5 mU/ml (A), cells expressing PH at a concentration of 1.5 mU/ml (B), and a mixture of cells expressing ToMO (1.5 mU/ml) and cells expressing PH (1.5 mU/ml) (C).

FIG. 5.

Kinetics of 3,4-DMC formation. Cells expressing PH were used at constant concentration (0.5 mU/ml), and the rate of 3,4-DMC formation was measured as a function of the concentration of cells expressing ToMO (⋄) in the presence of 40 μM o-xylene. The solid circle indicates the rate of 3,4-DMC production by cells coexpressing ToMO (1.17 mU/ml) and PH (0.5 mU/ml). The rate of 3,4-DMC production was measured by the continuous coupled assay with C2,3O as described in Materials and Methods. The inset shows the rate of 3,4-DMC formation as a function of the PH concentration at a constant concentration of ToMO (4.5 mU/ml).