A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin-dependent enzymes.

Area of Science:

• Biochemistry
• Environmental Microbiology
• Biocatalysis

Background:

• Halogenated phenols and nitrophenols are persistent environmental pollutants.
• The bacterium *Ralstonia pickettii* DTP0602 possesses the *had* operon with potential for degrading these toxins.
• HadA monooxygenase is known to perform dehalogenation and denitration but requires partner enzymes.

Purpose of the Study:

• To identify and characterize the flavin reductase and quinone reductase enzymes (HadB and HadX) that partner with HadA.
• To elucidate the functional roles and catalytic mechanisms of HadB and HadX in the *had* operon pathway.
• To reconstitute and evaluate the combined activity of HadA, HadB, and HadX for toxic compound degradation.

Main Methods:

• Overexpression and purification of HadB and HadX enzymes.
• Enzyme activity assays including transient kinetics and thermodynamics.
• Characterization of FMN and FAD cofactor utilization and electron transfer.
• Reconstitution of the complete HadA/HadB/HadX enzymatic cascade.

Main Results:

• HadB was identified as an FMN-dependent quinone reductase utilizing NADH and menadione.
• HadX was characterized as an FAD-bound flavin reductase that generates FADH^- for HadA via free diffusion.
• HadX demonstrated efficient NADH-mediated flavin reduction and could supply FADH^- to other monooxygenases.
• The combined HadA/HadB/HadX system effectively reconstituted a dehalogenation and denitration cascade.

Conclusions:

• HadB and HadX are crucial partner enzymes for HadA in the *had* operon.
• The identified enzymes enable the degradation of toxic halogenated and nitrophenol compounds.
• The reconstituted enzymatic cascade holds significant potential for future bioremediation strategies.