Substrate-assisted O2 activation in a cofactor-independent dioxygenase

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Most O2-activating enzymes require organic cofactors or metal ions for catalysis.
• Cofactor-independent oxygenases represent a distinct class of enzymes with unique catalytic mechanisms.

Purpose of the Study:

• To elucidate the O2 activation mechanism in a specific cofactor-independent dioxygenase.
• To understand how this enzyme, featuring an α/β-hydrolase fold, cleaves 2-alkyl-3-hydroxy-4(1H)-quinolones.

Main Methods:

• Chemical analysis of reaction intermediates.
• Electron paramagnetic resonance (EPR) spectroscopy to detect radical species.
• Characterization of enzyme kinetics and substrate interactions.

Main Results:

• O2 activation is substrate-assisted, involving the bound substrate anion.
• A single electron transfer from the substrate anion to O2 forms a radical pair.
• This radical pair recombines to generate a C2-peroxide intermediate.

Conclusions:

• Cofactor-independent oxygenases can function by utilizing the intrinsic reactivity of the substrate.
• Substrate activation to a (carb)anion by an active-site base is crucial for O2 interaction.
• This mechanism provides an alternative pathway for enzymatic oxygenation without external catalytic aids.