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 SkaAs essential catalytic role, with high-resolution liquid chromatography-tandem mass spectrometry identifying the key product 3-methyloxindole and 3-hydroxy-3-methyloxindole. Distribution studies revealed SkaA homologs predominantly in Actinobacteria, particularly Nocardia and Rhodococcus, indicating conserved 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 is a notorious, foul-smelling compound generated by the anaerobic breakdown of tryptophan, presenting substantial risks to both the environment and public health. Despite its prevalence, the enzymatic machinery initiating its biodegradation remains poorly characterized. Our study resolves this critical gap by identifying SkaA as a functionally validated enzyme catalyzing skatoles initial oxidation step. Distribution analyses revealed SkaA homologs predominantly in Actinobacteria, particularly Rhodococcus and Nocardia. Phylogenetic analysis positioned SkaA as a novel third subclass within Group E flavoprotein monooxygenases. These breakthroughs provide molecular tools for engineering targeted bioremediation solutions and new insights into the study of Group E FPMOs.