A novel two-domain laccase with a middle redox potential: physicochemical and structural properties

Abstract

The gene for a previously unexplored two-domain laccase was identified in the genome of the actinobacterium Streptomyces carpinensis VKM Ac-1300. A two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. The half-inactivation time of ScaSL at 80°C was 40 min. Laccase oxidized the non-phenolic organic compound ABTS at a maximum rate at pH 4.7; oxidized the phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. Laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was weakly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, laccase was completely inhibited by 100 mM sodium azide. Km and kcat of the enzyme for ABTS are 0.1 mM and 20 s-1, respectively. Km and kcat for 2,6-dimethoxyphenol are 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed the polymerization of humic acids and lignin. The redox potential of laccase was 0.472 ± 0.007 V. Thus, ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. The crystal structure of ScaSL was determined with a resolution of 2.35 Å. A comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.