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

Laccases are a class of multicopper oxidases that serve diverse functions across multiple industries. The laccase from Thermus thermophilus strain HB27 (TthMCO) is a thermostable enzyme characterized by a methionine-rich motif, proposed as a distinctive structural feature of laccases from Thermus, Meiothermus, and Aquifex. This motif in TthMCO and in the Multicopper oxidase from Aquifex aeolicus (McoA) partially occludes the T1 copper site solvent access (PDB entries 2XU9 and 6SYY, respectively). This structural feature, a β-hairpin in TthMCO, has been proposed to influence the catalytic activity and pH dependence of this enzyme. To address the knowledge gap, we generated two deletion variants: ∆1, removing the most mobile region (B-values >12 Å²), and ∆2, deleting the entire β-hairpin to mimic a fungal laccase architecture. This strategy allowed for direct comparison of structural integrity, catalytic parameters, and pH-dependent activity while maintaining the global fold. This work represents the first systematic combination of crystallography, kinetic analysis, and pH profiling applied to β-hairpin deletion mutants of TthMCO. Our results demonstrate that deletion of the β-hairpin expands the pH enzymatic activity range and improved the K_m values of the variants, although it negatively affected the overall reaction velocity. The findings described in this work reveal that the β-hairpin is critical for balancing substrate accessibility with electron-transfer efficiency, providing fundamental insights into the rational engineering of bacterial laccases related to TthMCO.