Comparative study of catalase-peroxidases (KatGs)

Area of Science:

• Biochemistry
• Enzymology
• Comparative Genomics

Background:

• Catalase-peroxidases (KatGs) are heme-containing enzymes with diverse catalytic functions.
• Understanding the variations and conserved properties of KatGs across different organisms is crucial for their biotechnological and therapeutic applications.

Purpose of the Study:

• To characterize and compare the biochemical properties of KatGs from seven distinct organisms.
• To elucidate the structure-function relationships and catalytic mechanisms of these enzymes.

Main Methods:

• Purification and characterization of KatGs from Archaeoglobus fulgidus, Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus, and Synechocystis PCC 6803.
• Enzyme kinetics assays were performed to determine turnover rates, pH optima, and inhibition constants (IC50) for various substrates and inhibitors.
• Analysis of kinetic parameters, including Michaelis constants (K(M)) and catalytic efficiencies (k(cat)), under different pH conditions.

Main Results:

• All seven KatGs displayed similar turnover rates for catalase (4900–15,900 s⁻¹) and peroxidase (8–25 s⁻¹) reactions.
• Similar pH optima were observed for peroxidase (4.25–5.0) and catalase (5.75) activities.
• High sensitivity to azide (IC50: 0.2–20 μM) and cyanide (IC50: 50–170 μM) was consistent across all enzymes.
• Michaelis constants for H₂O₂ in catalase reactions varied with pH, indicating protonation effects on distal histidine.
• Isoniazid activation rates were conserved but significantly slower (0.5 s⁻¹ for INH lyase, 0.002 s⁻¹ for isonicotinoyl-NAD synthase).
• NADH oxidase activity showed wider variation (10⁻⁴–10⁻² s⁻¹), with B. pseudomallei exhibiting the fastest rate.

Conclusions:

• KatGs from diverse organisms share conserved catalytic properties and inhibition profiles.
• Enzyme kinetics are influenced by pH-dependent protonation of key active site residues.
• The conserved, albeit slow, isoniazid activation suggests a critical role in anti-tubercular drug metabolism.
• Variations in NADH oxidase activity highlight potential specialized functions among different KatGs.