STRUCTURE ANALYSIS OF A NEW MARINE-DERIVED LACCASE

Laccases are multicopper oxidases used in many industrial and environmental applications. The search for new laccases resulted in the isolation of the marine-derived fungus Peniophora sp. CBMAI 1063, which is able to produce high amounts of these enzymes under optimized conditions. This fungus has a laccase multigene family and recently new genes were discovery. However, little is known about these genes and about the putative proteoforms that they codify. The present study aimed to perform an in silico structural characterization of the laccase proteoform codified by gene Pnh_Lcc2 from the marine-derived fungus Peniophora sp. CBMAI 1063. The gene was recently obtained from the genome annotation. Peptide chain prediction was performed using GeneRunner 5.0. The similarity analysis with other fungal laccases was carried out using BLASTP from NCBI database. The molecular weight was determined using ProtParam. The prediction of N-glycosylation sites was performed with the NetNGlyc 1.0. Tridimensional structure prediction was made through homology modeling using SWISS-MODEL (on-line version). The Pnh_Lcc2 gene showed 59 % of similarity with the laccase gene 2 from Trametes villosa. The putative laccase codified by Pnh_Lcc2 has approximately 58,14 kDa, discounting the glycosidic moiety. Five putative sites of N-glycosylation were found and the residues were identified as Asn159, Asn184, Asn349, Asn394 and Asn427. Two disulfide bridges may connect domains 1 and 3 (Cys103-Cys502), and domains 1 and 2 (Cys135-Cys222). On the active site, the T1 copper ion is located in the third domain and is coordinated by the residues His472 and Cys467 with angular geometry. Other histidine residue, His410, and two hydrophobic residues, Phe477 and Ile469, occur in the near surroundings of the T1 copper and may influence the redox potential of the enzyme. The trinuclear T2/(2)T3 copper center is located between domains 1 and 3 and copper ions are coordinated by eight residues of histidine: His32 (T2), His413 (T2), His129 (T3), His415 (T3), His466 (T3), His84 (T3), His127 (T3) and His 468 (T3). The results demonstrate that Pnh_Lcc2 encodes a new putative laccase, possibly with high redox potential and good stability. Considering little is known about marine-derived laccases structures, this study serves as a basis for further analysis such as crystallography and enzymatic kinetics.