Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Area of Science:

• Biochemistry
• Enzymology
• Environmental Microbiology

Background:

• Reductive dehalogenases are enzymes crucial for removing halogen atoms.
• Respiratory dehalogenases are oxygen-sensitive, hindering kinetic studies.
• Catabolic dehalogenases are oxygen-tolerant and attractive research targets.

Purpose of the Study:

• To achieve efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila.
• To characterize the enzyme's activity and potential for bioremediation.

Main Methods:

• Heterologous expression in Escherichia coli using maltose-binding protein and the btuCEDFB cobalamin uptake system.
• Enzyme activity assays for NADPH-dependent dehalogenation of phenolic compounds.
• Electron paramagnetic resonance (EPR) spectroscopy to study enzyme states.
• Growth studies using E. coli expressing the enzyme on a dehalogenated substrate.

Main Results:

• Efficient expression yielding high cobalamin occupancy and iron-sulfur cluster content.
• Enzyme efficiently dehalogenates brominated and iodinated phenols, including tetrabromobisphenol, under aerobic and anaerobic conditions.
• NADPH consumption is coupled to product formation; chlorinated compounds are competitive inhibitors.
• EPR spectroscopy indicates accumulation of Co(I)/(III) species.
• In vivo activity supports growth on a dehalogenated substrate, demonstrating bioremediation potential.

Conclusions:

• Efficient expression and characterization of a novel catabolic reductive dehalogenase.
• Demonstrated broad substrate range for dehalogenation of brominated and iodinated compounds.
• The enzyme shows significant promise for bioremediation applications.