Subtle changes in the active site architecture untangled overlapping substrate ranges and mechanistic differences of...

Area of Science:

• Biochemistry
• Environmental Microbiology
• Enzymology

Background:

• Reductive dehalogenases (RDases) are crucial iron-sulfur proteins in organohalide respiration.
• Understanding RDase mechanisms is vital for bioremediation of halogenated pollutants.

Purpose of the Study:

• To functionally analyze two distinct RDases, PceA and DcaA, from Desulfitobacterium species.
• To elucidate the differing catalytic mechanisms and active site roles in PceA and DcaA.

Main Methods:

• Heterologous protein production and functional characterization of recombinant PceA and DcaA.
• Site-directed mutagenesis to investigate active site residue roles (e.g., Tyr298, Trp118, Trp432, Thr294).
• In silico structural analysis of RDase active site cavities.

Main Results:

• DcaA efficiently converted 1,2-dichloroethane and 1,1,2-trichloroethane via dihaloelimination.
• PceA exclusively processed halogenated ethenes, lacking dihaloelimination activity.
• Mutation of Tyr298 abolished halogen substitution but not dihaloelimination, differentiating reaction mechanisms.
• Other mutations (W118F, W432F, T294V) affected DcaA function or substrate specificity.

Conclusions:

• PceA and DcaA exhibit divergent catalytic mechanisms despite high sequence identity.
• DcaA employs a mechanism for dihaloelimination that likely does not involve proton transfer.
• The active site residue Tyr298 is critical for halogen substitution but not dihaloelimination, and other residues influence substrate specificity.