Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Flavin-containing monooxygenases are crucial detoxifying enzymes with broad substrate selectivity.
• Their relaxed substrate selectivity is vital for metabolizing diverse xenobiotics and useful in synthetic chemistry.
• The molecular basis for this promiscuity in drug-metabolizing enzymes remains incompletely understood.

Purpose of the Study:

• To investigate the molecular basis of substrate selectivity in cyclohexanone monooxygenase (CHMO).
• To explore the role of active site residues in determining the substrate promiscuity of CHMO.
• To understand how active site architecture influences enzyme catalysis and substrate recognition.

Main Methods:

• Cumulative alanine mutagenesis was used to systematically alter active site residues in CHMO.
• Enzyme activity, protein folding, thermostability, and cofactor loading were assessed.
• Time-resolved kinetic studies and crystal structure determination were employed to analyze enzyme function and structure.

Main Results:

• Mutagenesis of up to eight active site residues, including phenylalanines, did not affect protein folding, stability, or cofactor binding.
• Enlarging the active site led to a linear decrease in catalytic activity without switching substrate preference.
• Crystal structure analysis revealed no significant conformational changes in the enzyme upon modification.

Conclusions:

• The studied CHMO may lack a distinct substrate selection mechanism, relying instead on its active site environment.
• The protein shell in promiscuous enzymes likely stabilizes reactive intermediates.
• Understanding enzyme promiscuity offers insights into both physiological detoxification and synthetic applications.