The β-hairpin effect: Structural insights into bacterial laccase function and stability

Area of Science:

• Enzymology
• Biochemistry
• Structural Biology

Background:

• Laccases are multicopper oxidases with industrial applications.
• The Thermus thermophilus HB27 laccase (TthMCO) features a unique methionine-rich motif and a β-hairpin structure.
• This β-hairpin partially blocks solvent access to the T1 copper site, potentially affecting enzyme activity.

Purpose of the Study:

• To investigate the role of the β-hairpin in TthMCO's catalytic activity and pH dependence.
• To engineer TthMCO variants by deleting the β-hairpin to mimic fungal laccase architecture.
• To understand the structural and functional implications of β-hairpin removal.

Main Methods:

• Generation of two deletion variants: ∆1 (removing mobile regions) and ∆2 (deleting the entire β-hairpin).
• Crystallography to assess structural integrity of variants.
• Kinetic analysis and pH profiling to determine catalytic parameters and pH-dependent activity.

Main Results:

• Deletion of the β-hairpin expanded the pH range for enzymatic activity.
• The K_m values of the deletion variants were improved, indicating better substrate binding.
• Overall reaction velocity (k_cat) was negatively affected by the β-hairpin deletion.

Conclusions:

• The β-hairpin in TthMCO is crucial for balancing substrate accessibility and electron-transfer efficiency.
• Deletion of the β-hairpin provides insights into rational engineering of bacterial laccases.
• This study offers fundamental understanding for optimizing laccase function in industrial settings.