Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.