Formatted Title
Characterization of Group-6 Soluble Di-Iron Monooxygenases for 1,4-Dioxane and Chlorinated Aliphatic Hydrocarbon Biodegradation
Background/Objectives
1,4-Dioxane (dioxane) metabolic biodegradation has been observed by soluble di-iron monooxygenases (SDIMOs) belonging to two groups, 5 and 6. Previous studies have been centered on group-5 tetrahydrofuran monooxygenases, while group-6 propane monooxygenases have been understudied. The limited understanding of group-6 propane monooxygenases hinders the development of effective and cost-efficient bioremediation strategies for dioxane mitigation. We identified a greater diversity of Group-6 propane monooxygenases. The phylogenetic differences among their α-subunits, in which the catalytic di-iron center is located, revealed functional distinction. In this present study, heterologous expression was employed to compare the functional characteristics of three representatives for group-6 propane monooxygenases. Three key functional parameters were characterized, including substrate range, enzyme kinetics with respect to dioxane, and inhibition kinetics by key cooccurring chlorinated aliphatic hydrocarbons (CAHs).
Approach/Activities
Amino acid sequences for α-subunits of the group-6 propane monooxygenases were aligned by ClustalX and the phylogenetic tree was generated in MEGA11. Three representatives were chosen for heterologous expression to compare functionality. Substrate range, kinetics, and inhibition kinetics were determined for each representative and used in the functional comparison of the three subgroups. After induction, transformants were exposed to different concentrations of dioxane with or without the presence of selected inhibitors (e.g., 1,1-DCE, 1,1,1-TCA, and TCE). The Michaelis-Menten and three inhibitory models were fitted with experimental data to evaluate the half saturation coefficients (Km), maximum degradation velocity (Vmax), and inhibition constant (KI).
Results/Lessons Learned
Phylogenetic analysis of the group-6 SDIMOs amino acid sequences in the highly conserved α-subunit revealed three distinct subclusters. Representatives from these three subclusters were expressed in the same heterologous host, Mycobacterium smegmatis mc2-155, which revealed functional distinctions. Substrate ranges were compared in four categories; cyclic and branched ethers, short-chain alkane/alkene gases, aromatic compounds, and CAHs; where we observed high degree of similarity among these subclusters. Furthermore, we expect, upon analysis of dioxane and inhibition kinetics, differences among the subclusters. This works will provide thorough understanding of dioxane degrading microbes and the responsible genes/enzymes, allowing us to better exploit dioxane remediation strategies at contaminated sites.