Tower Loop Engineering Converts a Low-Potential Bacterial Laccase into a High-Potential Laccase with Tunable Catalytic Activity

Abstract The reduction potential of type 1 copper (T1Cu) centers in laccases governs their catalytic efficiency and substrate scope, yet strategies to elevate low-potential bacterial laccases into the high-potential regime of fungal enzymes remain elusive. Here, we report a loop-engineering approach that enables large, tunable increases in T1Cu potential by redesigning the protein environment beyond the primary and secondary coordination spheres. Guided by structural comparison with fungal laccases, we introduce a “Tower loop” into the bacterial small laccase from Streptomyces coelicolor (SLAC), which shields the T1Cu site from solvent exposure and stabilizes favorable axial coordination. When combined with axial and secondary coordination-sphere mutations, this design elevates the T1Cu reduction potential from 367 mV to 708 mV vs. SHE, converting a low-potential bacterial laccase into a high-potential laccase. X-ray structural and spectroscopic analyses reveal that the introduced loop prevents solvent access and suppresses detrimental axial reorientation, providing a mechanistic basis for redox tuning. Kinetic studies further demonstrate a positive correlation between catalytic efficiency and the redox potential difference between the enzyme and substrate. These findings establish loop engineering as a general strategy for tuning redox-active metalloenzymes and provide a blueprint for designing high-performance biocatalysts for different applications from biomass valorization to bioelectrocatalysis.