Laccase belongs to the family of copper-containing oxidases. Eukaryotic laccases consist of three domains, while in bacteria in addition to the three-domain laccases there are two-domain (small laccases). Three-domain laccases have applications in the food industry, organic synthesis, cosmetics and medicine.Two-domain laccases are began to investigate recently, they have a higher thermostability and active in the alkaline pH range. These properties can be useful for different fields of industry. The objects of our studies are small laccases from Streptomyces species - Streptomyces griseoflavus and S. viridochromogenes. Despite the high sequence homology, the thermostability of proteins differs. Based on bioinformatic analysis we obtained mutant form of laccase from S. griseoflavus (SgSLmut) with substitutions of three methionines to leucines like in more thermostable laccase from S . viridochromogenes. Nevertheless biophysical investigations revealed that SgSLmut to be less thermostable comparing to wild type protein. Structural analysis of mutant form of laccase showed that occupancy of T2/T3 center by copper ions in mutant form is lesser than in a wild type protein. We suggested that reduced thermostability of SgSLmut could be result of the destabilization of functional trimer of laccase due to decrease of occupancy by copper ions of T2/T3 center. We have grown cells of strain-superproducent of SgSLmut with increased content of copper in the medium and obtain protein with almost full occupancy T2/T3 center by copper ions. Thermostability of such SgSLmut enhanced to the level of wild type protein.
laccase, mutagenesis, crystal structure, copper-containing center
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