Characterization of successive ortho-methyl oxidation of the priority pollutant 2,4-xylenol by a Rieske oxygenase system in Pseudomonas putida NCIMB 9866

The catabolism of the priority environmental pollutant 2,4-xylenol requires bacteria to oxidize its ortho-methyl group. Its exact biochemical mechanism is uncharacterized until now. In this study, we resolve key metabolic bottlenecks in Pseudomonas putida NCIMB 9866. We demonstrate that functionally redundant Rieske oxygenase systems ensure the robust ortho-methyl oxidation of 2,4-xylenol. On the para-methyl oxidation branch, PchA2 acts as the main aldehyde dehydrogenase. It shows significantly higher catalytic efficiency than the previously reported PchA. Additionally, we characterized PmmA1B1, a newly identified three-component Rieske oxygenase system responsible for ortho-methyl oxidation, which is supported by two redundant isoenzymes (PmmA2B2 and PmmA3B3), by recruiting the shared endogenous ferredoxin Orf05169. The PmmA1B1-Orf05169 system catalyzes the successive oxidation of the ortho-methyl group of 4-hydroxy-3-methylbenzoate to a carboxyl group, yielding the high-value pharmaceutical precursor 4-hydroxyisophthalate. Structural and kinetic data revealed that the active site architecture of PmmA1 enables efficient successive oxidation, distinguishing it from isoenzymes PmmA2B2 and PmmA3B3 that, when coupled with Orf05169, catalyze only incomplete oxidation. Overall, these results complete the 2,4-xylenol catabolic pathway by defining the enzymes required for both para- and ortho-methyl oxidation. They also reveal functional redundancy as an adaptive feature of microbial aromatic degradation and establish Rieske oxygenases as biocatalysts for converting methylated aromatic pollutants into value-added chemicals.