A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• The active-site loop of Bacillus subtilis oxalate decarboxylase (OxDC) influences Mn(II) reactivity.
• A conserved Arg/Thr hydrogen bond interaction is critical for OxDC function.

Purpose of the Study:

• To investigate the functional and structural impact of disrupting the conserved Arg/Thr hydrogen bond in OxDC via site-specific mutagenesis.
• To elucidate the role of this hydrogen bond in the enzyme's catalytic mechanism, particularly in proton-coupled electron transfer (PCET).

Main Methods:

• Site-specific mutagenesis was employed to substitute Threonine-165 with valine, creating the T165V OxDC variant.
• Catalytic activity assays were performed.
• Heavy-atom isotope effect measurements and X-ray crystallography were used to determine structural and mechanistic details.

Main Results:

• The T165V OxDC variant exhibited significantly impaired catalytic activity.
• X-ray crystallography revealed that the mutation affects the positioning of Glutamate-162, a key residue in the PCET step.
• The T165V variant showed reduced oxalate consumption per dioxygen molecule, suggesting a role for dioxygen as a reversible electron sink in PCET.

Conclusions:

• The conserved Arg/Thr hydrogen bond is essential for maintaining the correct conformation of Glu-162, facilitating the PCET step in OxDC.
• The findings suggest a more complex role for dioxygen in the OxDC reaction mechanism than previously thought, potentially involving reversible electron transfer.
• Understanding these mechanistic details is crucial for enzyme engineering and the development of novel biocatalysts.