Mutation of glutamic acid 103 of toluene o-xylene monooxygenase as a means to control the catabolic efficiency of a recombinant upper pathway for degradation of methylated aromatic compounds

Abstract

Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.

FIG. 1.

Proposed transformations of toluene (A) and o-xylene (B) catalyzed by ToMO and PH. The thickness of the arrows is roughly proportional to the relative abundance of each species. The possible catabolic fate of the hydroxylation products through the meta pathway is also shown. HMSD, 2-hydroxymuconic semialdehyde dehydrogenase which produces NADH; HMSH, 2-hydroxymuconic semialdehyde hydrolase which produces acetate (12); o-C, o-cresol; m-C, m-cresol; p-C, p-cresol.

FIG. 2.

Active site of ToMO A with a docked 2,4-DMP molecule (green). The carbon atoms of the residues that take part in the formation of the hypothetical subsites for methyl groups are red, yellow, green, magenta, orange, and cyan, whereas nitrogen and oxygen atoms of these residues are blue and light red, respectively. Iron ions are purple. All other atoms are white. The numbers of the residues are also shown. The color of the surface of each active site pocket corresponds to the color of the residues that form the cavity. Hydrogen atoms are shown only on the 2,4-DMP molecule.

FIG. 3.

Kinetics of 2,3-DMP (□), 3,4-DMP (▪), 3,4-DMC (○), and 4,5-DMC (•) production by a mixture of cells expressing ToMO (1.5 mU/ml) and PH (1.5 mU/ml) (A) and a mixture of cells expressing (E103G)-ToMO (1.5 mU/ml) and PH (1.5 mU/ml) (B) in the presence of 20 μM o-xylene.

FIG. 4.

Kinetics of 3,4-DMC formation. Cells expressing PH were used at a 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 (▪) or (E103G)-ToMO (□) in the presence of 40 μM o-xylene. 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 linearization of the data obtained by plotting rates (μM min⁻¹) as a function of the rate/[ToMO] ratio (μM min⁻¹/mU ml⁻¹). wt, wild type.