Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

Area of Science:

• Biochemistry
• Enzymology
• Organic Chemistry

Background:

• Antibiotic biosynthesis monooxygenases are cofactor-independent enzymes catalyzing O2-dependent oxidations.
• The mechanism by which these enzymes overcome the kinetic barrier for reactions between singlet substrates and triplet O2 remains unclear.
• A proposed model suggests these reactions proceed via a flavin-like mechanism, with the substrate substituting for a flavin cofactor.

Purpose of the Study:

• To investigate the proposed flavin-like mechanism of cofactor-independent monooxygenases using dithranol as a substrate for nogalamycin monooxygenase (NMO).
• To elucidate the role of conserved asparagine residues in substrate pKa suppression and enzymatic catalysis.
• To identify and characterize reaction intermediates, specifically a superoxide/substrate radical pair.

Main Methods:

• Monitoring of uncatalyzed and NMO-catalyzed dithranol oxidation reactions.
• pH-dependent kinetic studies.
• Site-directed mutagenesis to investigate the role of conserved asparagine residues.
• Detection of enzyme-bound superoxide during enzymatic turnover.
• Use of small molecule and enzymatic superoxide traps to assess reaction pathways.

Main Results:

• Dithranol oxidation, similar to flavin, showed increased rates at higher pH, with conserved asparagines suppressing substrate pKa.
• Enzymatic catalysis proceeded via an O2-dependent slow step, consistent with the flavoenzyme model.
• Enzyme-bound superoxide was detected, providing evidence for a superoxide/substrate radical pair intermediate.
• NMO was found to accelerate the formation and direct the recombination of this radical pair, unlike uncatalyzed reactions.

Conclusions:

• Nogalamycin monooxygenase (NMO) employs a flavin-like catalytic strategy involving a superoxide/substrate radical pair intermediate.
• Conserved asparagine residues play a crucial role in substrate activation and enzymatic catalysis.
• These findings provide insights into cofactor-independent oxygenation mechanisms, contrasting with other known enzyme systems.