Estudo in silico da atividade catalítica da lipase de Fusarium oxysporum f. sp. tracheiphilum na bioconversão do diterpeno annonalida e seus derivados acilados

Abstract

Previous studies carried out in our research group revealed the enzymatic potential of the filamentous fungus Fusarium oxysporum f. sp. tracheiphilum (strain UFCM 0089) in the biotransformation of the natural product annonalide (1), isolated from the roots of the plant species Casimirella ampla, and its O-acyl semi-synthetic derivatives (4-7) in the side chain. The formation of the products and their proportions, as well as the enzymatic activity of the fungus, were dependent on the presence of the acyl group in the molecule, besides the size of the lipophilic chain. In all cases, fungal lipase activity played an important role on the product formation. Thus, the in silico study on the activity of lipase from the fungus F. oxysporum in the bioconversion of 1 and its O-acylated derivatives (4-7) was carried out to help the understanding of the experimental results. Using the primary sequence of the lipase from the fungus F. oxysporum and the crystallographic coordinates of the lipase from Thermomyces lanuginosus (PDB ID: 1EIN), molecular homology modeling techniques were used to obtain the three-dimensional structure of the lipase from F. oxysporum. After obtaining a model, a simulation of energy minimization in a water tank was performed, in order to improve the contacts and geometries of the modeled structure. The final modeled conformation was used in the molecular anchoring steps, together with the molecules (substrates) to be investigated. Among the obtained results, it was possible to characterize the enzyme-substrate interaction mode, selecting the poses that followed the NAC criteria. Finally, the selected complexes (enzyme-substrate) went through the induced coupling process, which allowed the complementary adjustment between the enzyme and substrate conformations, mimicking the process observed in vitro. The theoretical results corroborate the experimental ones, showing that the increase in the carbon chain of the acyl group in the side chain of the investigated substrates (1, 4-7) increases the lipase activity of the fungus F. oxysporum. Initially, the molecular anchoring simulations allowed us to identify the conformations most susceptible to the attack of the enzyme's catalytic serine on carbon 19 of the lactone ring of annonalide (1) and carbon 21 of the side chain of the O-acylated derivatives (4-7). Then, the induced coupling simulations confirmed the stability of some of the conformations obtained in the molecular docking, revealing the Lig1_conf1, Lig5_conf1, Lig6_conf1 and Lig7_conf1 conformations as being the most stable. The Lig4_conf1 conformation showed low stability compared to the other substrates. The MM-PBSA calculations allowed to estimate the affinity of the biomolecular complexes and showed that the increase in the acyl side chain of the substrate increased the lipase activity. All these results corroborated the experimental biotransformation of substrates by growing cells of the fungus.