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Laccases are multicopper oxidases that catalyze oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. The physicochemical and catalytic properties of two new fungal laccases from basidiomycetes Antrodiella faginea (AfL) and Steccherinum murashkinskyi (SmL) with middle redox potential of the T1 copper site were studied. The X-ray structures of AfL and SmL were solved at 1.75 Å and 0.95 Å, respectively. The oxidized state of copper ions in the active site was observed in AfL structure, while the mixture of oxidized and reduced states was observed in SmL structure. These oxidized and reduced states relate to the position of copper ions, their coordination, and nature and position of oxygen ligands. Comparative analysis of the T1 site environment of laccases with known structure allowed us to highlight the six types of the secondary coordination sphere of the T1 copper. The solvent accessible surface area of the conservative region of the secondary coordination sphere of the T1 copper correlates with its the redox potential. It was shown that the laccase classification by the structure of the T1 copper secondary coordination sphere is in agreement to ecophysiological behavior of laccase producing fungi. [ABSTRACT FROM AUTHOR]

• The ESI FT-ICR MS was applied for studying kraft lignin modification by T. hirsuta. • The main patterns of lignin modification were determined. • The groups of systematically degraded and released compounds were identified. • The main enzymes used by the fungus were versatile and manganese peroxidases. • The order of peroxidase secretion was connected with lignin modification patterns. The process of kraft lignin modification by the white-rot fungus Trametes hirsuta was investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS), and groups of systematically changing compounds were delineated. In the course of cultivation, fungus tended to degrade progressively more reduced compounds and produced more oxidized ones. However, this process was not gradual – the substantial discontinuity was observed between 6th and 10th days of cultivation. Simultaneously, the secretion of ligninolytic peroxidases by the fungus was changing in a cascade manner – new isoenzymes were added to the mixture of the already secreted ones, and once new isoenzyme appeared both its relative quantity and number of isoforms increased as cultivation proceeded. It was proposed, that the later secreted peroxidases (MnP7 and MnP1) possess higher substrate affinity for some phenolic compounds and act in more specialized manner than the early secreted ones (MnP5 and VP2). [ABSTRACT FROM AUTHOR]

Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is an enzyme that has been studied for over 100 y and is present in virtually all fungi. As increasing numbers of fungal genomes have been sequenced, it has become apparent that the laccase genes in white-rot fungi commonly form multigene families consisting of many nonallelic genes. Although a number of reports focussing on laccase gene expression in different fungal species were published over the decades, the fundamental questions of why fungi need such a redundant array of genes and how they manage this array to perform biological function(s) remain far from answered. In this article, we present a comprehensive study of the transcription of the whole Trametes hirsuta laccase multigene family under different conditions, including exposure to different nutritional factors such as nitrogen sources (organic and inorganic) and concentrations of nitrogen and carbon in the culture medium; in different growth phases (lag phase and stationary phase); and in the presence of different inducer agents (water-soluble lignin, bromocresol green dye, p -coumaric acid, ferulic acid, guaiacol, vanillin, veratryl alcohol, vanillic acid and syringic acid). Our findings are discussed in the context of the evolution of the laccase multigene family, and the presence of transcription-level subfunctionalization is highlighted. [ABSTRACT FROM AUTHOR]

Laccases, blue copper-containing oxidases, ≿an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072 , an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed. [ABSTRACT FROM AUTHOR]