Bidentate Substrate Binding Mode in Oxalate Decarboxylase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Bioinorganic chemistry

Background:

• Oxalate decarboxylase (EC 4.1.1.2) is an Mn- and O2-dependent enzyme crucial for oxalate metabolism.
• Existing models propose monodentate substrate binding to facilitate dioxygen access, despite oxalate's preference for bidentate coordination.

Purpose of the Study:

• To investigate the substrate binding mode of oxalate decarboxylase using experimental and computational methods.
• To revise mechanistic hypotheses regarding substrate binding and dioxygen interaction in oxalate decarboxylase.

Main Methods:

• X-band 13C-electron nuclear double resonance (ENDOR) spectroscopy on 13C-labeled oxalate.
• Domain-based local pair natural orbital coupled cluster singles and doubles (DLPNO-CCSD) calculations.
• Density functional theory (DFT) geometry optimization of active site models.

Main Results:

• ENDOR experiments demonstrate bidentate (κO, κO') binding of oxalate to the active site Mn(II) ion across different conditions and enzyme variants.
• Computational methods predict ENDOR spectra consistent with bidentate binding and show it is energetically favored over monodentate binding.
• The energetically preferred bidentate binding reconciles experimental data and challenges prior assumptions about dioxygen binding.

Conclusions:

• Oxalate decarboxylase binds its substrate, oxalate, in a bidentate fashion to the active site Mn(II) ion.
• This finding necessitates a revision of existing mechanistic models, particularly concerning the role and binding site of dioxygen.
• The results support a revised mechanism involving long-range electron transfer for substrate activation.