Understanding Lignin Oxidation by a Two-Domain Multicopper Oxidase from Cellvibrio japonicus.

Area of Science:

• Biochemistry and enzymology
• Molecular biology
• Microbial metabolism

Background:

• Bacterial two-domain multicopper oxidases (2dMCOs) are structurally distinct from fungal laccases.
• Their biochemical properties and roles in lignin conversion are poorly understood.
• Type B 2dMCOs possess a T1 active site within a trimer center tunnel.

Purpose of the Study:

• To comprehensively characterize the biochemical properties of a type B 2dMCO from Cellvibrio japonicus (CjMCO).
• To investigate the relationship between CjMCO's structure and its lignin oxidation activity.
• To explore the potential of CjMCO in lignin modification and understand its physiological roles.

Main Methods:

• Biochemical assays to determine redox potential and turnover number.
• Stopped-flow UV-vis spectroscopy to analyze enzyme kinetics and reoxidation rates.
• Oxidation of lignin model compounds and oligomeric lignin to assess activity and product profiles.

Main Results:

• CjMCO exhibits a T1 copper redox potential of 537 mV but a low turnover number (0.4 s⁻¹), ~1000-fold lower than fungal laccases.
• Slow reoxidation by O₂ was identified as a limiting factor for CjMCO turnover.
• CjMCO oxidized lignin model compounds and oligomeric lignin, causing both coupling and bond cleavage, with activity influenced by pH, buffer, and ionic strength.

Conclusions:

• CjMCO functions as a laccase, oxidizing phenolic lignin moieties.
• Its slow catalytic rates and trimeric structure suggest limited efficiency in degrading lignin polymers in vivo.
• This research expands understanding of bacterial laccase diversity and suggests potential roles for type B 2dMCOs beyond lignin degradation.