Most of the currently known fungal laccases show their maximum activity under acidic environmental conditions. It is known that a decrease in the activity of a typical laccase at neutral or alkaline pH values is the result of an increase in the binding of the hydroxide anion to the T2/T3 copper center, which prevents the transfer of an electron from the T1 Cu to the trinuclear copper center. However, evolutionary pressure has resolved the existing limitations in the catalytic mechanism of laccase, allowing such enzymes to be functionally active under neutral/alkaline pH conditions, thereby giving fungi an advantage for their survival. Combined molecular and biochemical studies, homological modeling, calculation of the electrostatic potential on the Connolly surface at pH 5.0 and 7.0, and structural analysis of the novel alkaliphilic laccase of Myrothecium roridum VKM F‐3565 and alkaliphilic and acidophilic fungal laccases with a known structure allowed a new intramolecular channel near the one of the catalytic aspartate residues at T2‐copper atom to be found. The amino acid residues of alkaliphilic laccases forming this channel can presumably serve as proton donors for catalytic aspartates under neutral conditions, thus ensuring proper functioning. For the first time for ascomycetous laccases, the production of new trimeric products of phenylpropanoid condensation under neutral conditions has been shown, which could have a potential for use in pharmacology.

A new unusual biotechnologically valuable laccase from the ascomycete Myrothecium (Paramyrothecium) roridum VKM F-3565 with a molecular weight of about 80 kDa, which exhibits the maximum oxidative activity against a number of phenolic compounds and phenylpropanoids under neutral-alkaline conditions (pH 6.5-7.8), was isolated and characterized. For the first time for laccases of ascomycetes, the production of new trimeric products of ferulic acid and coniferyl alcohol condensation has been shown, which presumably have a potential for use in pharmacology. The gene encoding the unusual alkaliphilic laccase has been isolated for the first time from fungi of the Myrothecium ( Paramyrothecium) genus. The high phylogenetic relationship between the M. roridum VKM F-3565 laccase gene and genes presumably encoding the laccases of ascomycetes of the genera of Colletotrichum, Verticillium, and Fusarium was shown. Homological modeling, calculation of the electrostatic potential on the molecular surface at pH5.0 and 7.0, and structural analysis of the studied laccase as well as the fungal alkaliphilic and acidophilic laccases with a known structure allowed finding a new intramolecular channel, different from the other known internal channels, located near the catalytic aspartate residue at the T2-copper atom. The amino acid residues of alkaliphilic laccases forming the found channel presumably serve as proton donors for the catalytic aspartate, ensuring its proper functioning.

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