A novel and evolutionarily distinct flavoprotein monooxygenase drives skatole degradation in Rhodococcus

Skatole (3-methylindole), a persistent and toxic N-heterocyclic aromatic pollutant, poses significant bioremediation challenges due to poorly defined biodegradation processes. This study systematically investigated the genetic determinants and metabolic mechanisms of skatole degradation in the environmentally versatile Gram-positive genus Rhodococcus. We demonstrated that skatole degradation was a broadly conserved trait across diverse Rhodococcus species, including R. aetherivorans, R. pyridinivorans, R. ruber, and R. qingshengii. Genomic and functional analyses uncovered a novel flavoprotein monooxygenase (FPMO) SkaA as the initial catalyst for skatole catabolism. Heterologous expression in Escherichia coli confirmed SkaA's essential catalytic role, with high-resolution liquid chromatography-tandem mass spectrometry identifying the key products 3-methyloxindole and 3-hydroxy-3-methyloxindole. Distribution studies revealed SkaA homologs predominantly in Actinobacteria, particularly Nocardia and Rhodococcus, indicating metabolic capacity for skatole transformation within this phylum. Notably, Rhodococcus strains lacking the skaA gene also retained skatole degradation activity, implying the existence of alternative genetic determinants. Crucially, phylogenetic analysis positioned SkaA as a distinct subclass within Group E FPMOs, exhibiting ≤40% sequence identity to reported styrene/indole oxygenases. The phylogenetic segregation of skatole, indole, and styrene monooxygenases provides a predictive framework for functional annotation of Group E FPMOs. Our findings elucidate a novel skatole transformation pathway, establish Rhodococcus as environmentally versatile biocatalysts, and provide new insights into the study of Group E FPMOs.IMPORTANCESkatole, a notorious malodorous compound, is mainly produced via the microbial anaerobic degradation of tryptophan. Its presence raises significant environmental and public health concerns. Yet, the enzymes responsible for its biodegradation remain poorly characterized. This study demonstrates that SkaA catalyzes the initial oxidation step in skatole metabolism. Distribution analysis reveals that SkaA homologs are predominantly in Actinobacteria, especially within the genera Rhodococcus and Nocardia. Phylogenetic analysis positions SkaA in a novel subclass within Group E flavoprotein monooxygenases (FPMOs). These findings not only provide crucial insights into skatole metabolism but also expand our understanding of the functional diversity of Group E FPMOs.