Distal design improves thermostability and enzyme activity of type III tyrosinase from Nitrosospira

Tyrosinase (TYR), a copper-containing oxidase pivotal in melanin synthesis, is widely distributed across animals, plants, and microorganisms. Despite its significant potential in biotechnology and industry, its practical application is hampered by limitations such as low catalytic efficiency and poor stability. To address these constraints, a highly active type III TYR from Nitrosospira (Sp2) was identified through systematic genomic mining in this study. Based on the structural features of type III TYR, two C-terminal truncated mutants were constructed. Among them, the truncated mutant TYR-Sp2-276 achieved an enzyme activity of 317 U/mg, which is a 15% increase compared to the wild-type. Subsequent protein engineering adopted a distal design strategy, which rationally targets residues remote from the catalytic center coupled with computational simulations to construct a combinatorial mutant library. The combinatorial mutant TYR-Sp2-276-G73A/M106D/Q152A/M231P exhibited a 2.37-fold enhancement in enzymatic activity, reaching 654 U/mg. Its melting temperature (T_m) increased by 4.59 °C, while the k_cat value showed a 2.55-fold improvement. Structural predictions from AlphaFold 3 and molecular docking indicate that changes in structural rigidity and microscopic interactions such as hydrogen bonding may be responsible for the enhancement of its catalytic activity and thermal stability. This work demonstrates that rational distal design is an effective strategy for optimizing enzyme properties, offering valuable insights for engineering industrially relevant microbial enzymes.