Substrate promiscuity and active site differences in gentisate 1,2-dioxygenases: electron paramagnetic resonance...

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Gentisate 1,2-dioxygenases (GDOs) are non-heme iron enzymes oxidizing dihydroxylated aromatic compounds.
• Salicylate 1,2-dioxygenase (SDO), a GDO family member, oxidizes monohydroxylated substrates, exhibiting broader substrate specificity.
• The basis for substrate specificity differences between GDOs and SDOs remains unclear despite sequence similarity.

Purpose of the Study:

• To investigate the structural and mechanistic basis for substrate specificity differences between GDOs and SDOs.
• To compare the ferrous-nitrosyl complexes of SDO and a bacterial GDO (GDO_Cg) using electron paramagnetic resonance (EPR).
• To elucidate the role of specific residues in determining substrate binding and catalytic activity.

Main Methods:

• Electron paramagnetic resonance (EPR) spectroscopy was employed.
• Ferrous-nitrosyl complexes of SDO and GDO_Cg were prepared and analyzed.
• A variant GDO_Cg(A112G) was studied to assess the impact of mutation on enzyme mechanism.

Main Results:

• EPR spectra revealed distinct substrate binding modes and metal cofactor coordination geometries between SDO and GDO_Cg.
• Substrate binding increased the symmetry of the Fe(II)-NO complex in SDO, whereas it decreased symmetry in GDO_Cg.
• The A112G mutation in GDO_Cg did not alter the ternary complex structure, despite enabling salicylate oxidation.

Conclusions:

• Significant differences exist in the active site environment and substrate interaction between GDOs and SDOs.
• The observed variations in metal coordination and substrate binding likely contribute to the differential substrate specificities.
• The A112G mutation highlights that altered substrate oxidation can occur without significant changes to the Fe-substrate-O₂ intermediate structure.