-STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CATALYTIC SITE IN FUNGAL LACCASES OF BIOTECHNOLOGICAL INTEREST: MOLECULAR KEYS TO LIGAND COUPLING

AYALA SCHIMPF AR; GAMARRA MD; FONSECA MI; ZAPATA PD

Congreso; XVII Congreso Argentino de Microbiología General; 2022

White-rot basidiomycete laccases are capable of catalyzing the oxidation of a wide range of substrates,resulting in excellent biotechnological candidates for biodegradation and monitoring ofenvironmental compounds; that is why it is necessary to delve into the particularities of the interactionbetween the active site of these enzymes with the different substrates of interest, making use ofreported models with co-crystallized substrates such as ABTS. In this sense, Molecular Dockingsimulations allow protein-ligand coupling to be predicted, and it has been reported that by havinginformation on ideal sites (pharmacophoric) of the receptor, docking is considerably improved interms of precision and energy. The objective of the work was to characterize the catalytic site of threelaccases belonging to the fungus Pleurotus pulmonarius LBM 105 from the analysis of the interactionsites and residues involved in the conformations resulting from the coupling with ABTS. The threelaccases PplacI, Ppul2 and PpAS3 (GenBank Acc.No. OK403251.1, OK403252.1, OK403253.1) werehomology modeled by Phyre2, using the laccase structure of Trametes versicolor (PDBID: 1GYC) astemplate. To evaluate the validity and quality of the structures obtained, the MolProbity server wasused. Subsequently, the models were contrasted with the co-crystallized structure of the Bacillussubtilis laccase (PDBID: 3ZDW) as a reference for the characterization of the amino acid environmentof each model. Then, the calculation of the pharmacophoric sites was carried out using from theideal_sites.py AutoDockTools module available inside the MGLTools1.5.7 package. Finally, an aminoacid mask of the potential active site obtained in the calculations using CASTp 3.0 and DoGSiteScorerwas defined. The results were compared against the 3ZDW crystal (containing co-crystallized ABTS)expressed in RMSD values for each case. The molecular docking study was performed using AutoDock4.2. The MolProbity score obtained was 1.58 (PplacI), 1.57 (Ppul2), and 2.28 (lacIII). All laccases hadcorrespondence with the crystallized ABTS binding site, showing the same electron acceptor sitepresent in interaction with one of the histidines that coordinates Cu; PplacI and Ppul2 presented ahydrophobic region within the pocket very close to the T1 site indicating a strong interaction with ahydrophobic or aromatic group of the ligand while PpAS3 revealed other acceptor sites, with a polarenvironment capable of stabilizing the binding of polar groups. Using these biases in coupling withABTS generated structural conformations of high affinity energy and greater similarity to thecrystallized structure: PplacI (-5.42 kcal/mol-1), Ppul2 (-9.92 kcal/mol-1) and PpAS3 (-6.56 kcal/mol-1)with an RMSD of 5.93 Å, 5.81 Å and 5.79 Å respectively. These results lay the groundwork for theidentification of affinity-determining structural features in these fungal laccases in order to improvetheir substrate-binding capacity.