Divergent Catalytic Strategies of Group-6 Propane Monooxygenases in Propane-Stimulated 1,4-Dioxane Biodegradation

Abstract Group-6 propane monooxygenases (PRMs) catalyze the initial oxidation of 1,4-dioxane (dioxane), a widespread contaminant of emerging concern. Because their expression can be induced by propane, an inexpensive and readily deliverable substrate, PRM-containing microorganisms offer a distinct advantage for propane-based biostimulation strategies targeting dioxane removal. However, functional differences among PRMs from the three major phylogenetic clusters remain poorly understood. Here, three PRMs from Mycobacterium dioxanotrophicus PH-06, Mycobacterium marinum E11, and Mycobacterium sp. ENV421, representing phylogenetic clusters, PRM-I, PRM-II, and PRM-III, respectively, were heterologously expressed in Mycolicibacterium smegmatis mc2155. ENV421 PRM-III exhibited low substrate affinity but high catalytic capacity toward both dioxane and propane, and moderate resistance to chlorinated solvent inhibition. In contrast, E11 PRM-II displayed high affinity but lower capacity and greater inhibitor sensitivity. PH-06 PRM-I showed comparatively stronger activity toward dioxane than propane relative to the other PRMs. Protein structure analysis suggested that variation in substrate affinity and catalytic turnover is consistent with differences in active-site pocket hydrophobicity, rigidity, and catalytic geometry. Phylogenetic and compositional analyses further suggested horizontal transfer in Clusters I and III. Together, these results highlight substantial functional diversity among group-6 PRMs and offer mechanistic insights for optimizing microbial populations for effective propane-stimulated bioremediation of dioxane.