Methylhydroxylase encoded by mchAB gene is involved in methyl oxidation of m-cresol via Comamonas thiooxydans CHJ601

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 K m value of m -cresol (48.74 ± 5.02 μM) for MchAB was much lower than that of o -cresol (747.80 ± 53.34 μM) and p -cresol (541.90 ± 41.43 μM). 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-hydroxybenzaldehyde 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.