Regiospecificity of two multicomponent monooxygenases from Pseudomonas stutzeri OX1: molecular basis for catabolic adaptation of this microorganism to methylated aromatic compounds.
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.
Cafaro
Valeria
V
Dipartimento di Biologia strutturale e funzionale, Università di Napoli Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia, 80126 Naples, Italy.
Notomista
Eugenio
E
Capasso
Paola
P
Di Donato
Alberto
A
D000222:Adaptation, Physiological; D001673:Biodegradation, Environmental; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006841:Hydrocarbons, Aromatic / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism; D014992:Xylenes / Q000378:metabolism
D006841:Hydrocarbons, Aromatic; D009097:Multienzyme Complexes; D014992:Xylenes; D014050:Toluene; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C114360:toluene 2-xylene monooxygenase; C022336:phenol 2-monooxygenase