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.

Isolation and molecular characterization of a novel <i>pseudomonas putida</i> strain capable of degrading organophosphate and aromatic compounds

“…While this may certainly result from incompleteness of the available POXN01 genome sequence, there is a possibility that the toluene ring fission is initiated by phenol/benzene hydroxylase. Indeed, this enzyme exhibits quite a relaxed specificity towards their aromatic hydrocarbon substrates, and can oxidize toluene albeit at a lower rate than phenol and benzene [63]. Similarly, it might hydroxylate p-nitrophenol released during paraoxon hydrolysis, thus making it susceptible to dioxigenase-mediated ring cleavage.…”

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

“…Phenol hydroxylases, for the most part, are ortho-hydroxylating enzymes, whereas toluene monooxygenases hydroxylate in the para position (58,74). PHH and ToMOH from P. stutzeri OX1, however, have relaxed regiospecificities and typically afford 70% ocresol and 45% p-cresol, respectively (20,75,76). For alternative substrates, PH and ToMO perform highly regiospecific reactions.…”

“…For alternative substrates, PH and ToMO perform highly regiospecific reactions. For example, PH and ToMO yield 96% 3-methylcatechol and 95% 4-methylcatechol, respectively, when m-cresol is used as the substrate (76,77). The hydrophobic residues that line the active site pockets of PHH and ToMOH are mostly conserved among their specific subfamilies (Table 3) (3).…”

Molecular Determinants of the Regioselectivity of Toluene/ o -Xylene Monooxygenase from Pseudomonas sp. Strain OX1

“…For example, TMOs and PHs perform two consecutive hydroxylation reactions with aromatic rings, but usually TMOs are more efficient in the first hydroxylation step, whereas PHs are more efficient in the second (3,5). Moreover, each TMO and PH shows its own characteristic regioselectivity.…”