Investigations of accessibility of T2/T3 copper center of two-domain laccase from streptomyces griseoflavus ac-993

Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3a and Cu3ß), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Streptomyces griseoflavus Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His 165 is a “gateway” at the O2-tunnel leading from solvent to the Cu3ß of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His 157 that belongs to the first coordination sphere of Cu3a. ; This research was funded by the Russian Foundation for Basic Research (Grant # 18-04-00270a), the RAS Program for Molecular and Cell Biology and Post-Genomic Technologies. Works on proteins crystallization was supported by Russian Science Foundation (grant No. 17-14-01207).