The compound m-cresol (meta-cresol) is an important representative of alkylphenol pollutants with high toxicity and potential carcinogenicity. The genes and enzymes associated with the m-cresol pathway have not yet been well elucidated. In this study, Comamonas thiooxydans CHJ601 was isolated from industrial wastewater and cultured utilizing m-cresol as the sole source of carbon and energy. m-Cresol catabolism begins with the oxidation of its methyl group in strain CHJ601. Genome sequencing and heterologous expression of key genes revealed its detailed catabolic pathway. m-Cresol methylhydroxylase (MchAB) catalyzed o-cresol (ortho-cresol), m-cresol, and p-cresol (para-cresol) in the presence of NADH and FAD. The Km value of m-cresol (48.74 +/- 5.02 mu M) for MchAB was much lower than that of o-cresol (747.80 +/- 53.34 mu M) and p-cresol (541.90 +/- 41.43 mu M). Molecular docking analysis showed that the binding affinities of MchAB against m-cresol with two hydrogen bonds are higher than p-cresol methylhydroxylase with one. The optimal substrate of MchAB was m-cresol, which can be catalyzed into 3-hydroxybenzyl alcohol and then oxidized to 3-hydroxybenzaldehyde. The mchC gene encoded 3-hydroxyben-zaldehyde dehydrogenase, which catalyzes the oxidation of 3-hydroxybenzaldehyde to 3-hydroxybenzoate. The results indicated that MchAB and MchC are the first characterized enzymes to catalyze the oxidation of the meta- methyl group of m-cresol in the strain CHJ601.
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