Study On Structural Transformation Of Fungal Laccase Based On Catalysis Mechanism And Their Potential For Degrade Organophosphorus Poisons

ABSTRACTObjectives:1 Study the interaction between enzyme and ligand of fungal laccase complex and further reveal the catalysis mechanism.2 Analysis the regularity of laccase for degrade organophosphate poisons and exploring the catalysis mechanism.3 Design fungal laccase and predict the effect of these mutant laccases for degrade organophosphorus poisons. Then obtain the mutation laccase through experiments which has good effect by theory predicts.Methods:1 Study the interaction between enzyme and ligand of five fungal laccases in crystalline and amorphous state by using molecular modeling technique and interaction energy optimization method, and further reveal the catalysis mechanism at the molecular level.2 Analysis the regularity of laccase for degrade organophosphate poisons based on the analysis of available experimental studies. Then exploring the interaction between that two by use molecular docking technique.3 Analysis of the strategy for design fungal laccase based on mechanism of laccase-mediator system and proposed the mutation direction. After select the appropriate transformation of target laccase, we bulid these models of mutation enzyme by using homology modeling technique.4 Study the interaction between mutant laccase and VX by using molecular docking technique, and predicte their effects by analyze binding mode and affinity between that two.5 Obtain the mutation laccase through Pichia expression system which has good effect by theory predicts.Results:1 In the structure of 1KYA, these amino acid residues which have hydrophobic interaction with substrate are Phe162, Leu164, Phe265, Pro391 and Gly392. While in the structure of 2HRG, they are Val162, Phe331, Pro390 and Gly391. In the structure of 3FU7, 3FU8 and 3FU9, the ligand has hydrophobic interaction with Ala191, Pro192, Phe371, Phe427 and Leu429 in the active site. In addition, the “electron-rich” group in substrate structure is close to a conserved histidine, and always formate a hydrogen-bond.2 Fungal laccase-mediator system can degrade organophosphate poisons which have a signature structure of class-V nerve agents effectively. The laccase is all from white rot fungi. The mediator could enhance oxidation capacity of laccase and reduce steric hindrance between that two. In addition, The nitrogen atom of these organophosphate poisons is likely to be an important factor in degradation: when there are two isopropyl substituents, the efficiency is significantly higher than two ethyl substituents.3 MOE-Dock module is used to explore the interaction between fungal laccase and organophosphate poisons. While explore the optimal conditions for docking, we add charge and hydrogen to enzyme and substrate in Amber99 forcefield or MMFF94 x forcefield respectively, continues for docking in MMFF94 x forcefield, select Triangle Matcher(placement function), London d G(scoring function), Forcefield(optimization function), and assess the results based on binding free energy. The result could demonstrate the interaction between fungal laccases and substrate.4 While bind to the active site of fungal laccase, the organophosphate poisons have hydrophobic interaction with Phe162, Pro391, Gly392 and Ala393 in the active site. The nitrogen atom in organophosphate poisons is close to a conserved histidine. The substrate would formate a hydrogen-bond with Gly392.5 In order to enhance the capacity of laccase to degrade toxic materials, we propose an idea: transform several amino acids in the active site and enhance the binding affinity between enzyme and organophosphate poisons. Trametes versicolor laccase is widely distributed in nature, and it achieve good results in degradate organophosphate poisons. We believe that it is a good candidator for our transformation object.6 MOE-Homology Model module is used to establish mutant laccase models. While explore the optimal conditions to do this work, we select Amber99(forcefield function), GB/VI(scoring function), and take care of the “outside” amino acids found in ramachandran plot.7 We build F162 A mutation, D206 A mutant, F265 A mutant, F332 A mutation, P391 A mutation, P391 L mutation and F162A/F332 A mutation structural models. The “outside” amino acids found in ramachandran plots are all outside of avtive site, and the number does not exceed 0.6% in total. Furthermore, there is no significantly change on the shape of active site. Their active site are all enlarge besides D206 A mutant and P391 L mutation. The F265 A mutant, F332 A mutation and F162A/F332 A mutation would even enlarge the “door” of active site.8 The binding free energy of F265 A mutation, F332 A mutation, P391 A mutation and P391 L mutation with VX is reduced, while increased for F162 A mutation, D206 A mutation and F162A/F332 A mutation. The P391 A mutation has a biggest change(almost 10% reduced).9 The interaction between mutation laccases and organophosphate poisons are just like the mode before mutation design: the substrare have hydrophobic interaction with Phe162, Pro391, Gly392 and Ala393 in the avtive site. The nitrogen atom in substrate is close to a conserved histidine. The substrate would formate a hydrogen-bond with Gly392.10 In the event of F162 A mutation, F332 A mutant, P391 A mutation and F162A/F332 A mutation, the distance between nitrogen atoms of VX and NE2 atom of histidine are dropped from 6.67? to 4.63?, 4.56? and 4.45? and 4.51? greatly.11 We obtain 4ml of the P391 A mutation laccase(0.24mg/ml) which has good effect by theory predicts through Pichia GS115 expression system.Conclusions:1 Hydrophobic interaction is an important factor in affecting substrate and oxidation products alternating combinate with laccase or release from it. When binding to the activite site, the “electron-rich” group in substrate structure always formate a hydrogen-bond with a conserved histidine. This hydrogen-bond may enhance the efficiency of electron transfer from substrate to laccase, and will improve catalytic efficiency.2 The interaction between fungal laccase and organophosphate poisons are almost alike: the substrate have hydrophobic interaction with Phe162, Pro391, Gly392 and Ala393, and it is the main binding force between that two; The N-terminal of substrate is embed to the active site and close to a conserved histidine, which would improve the efficiency of electron transfer from substrate to laccase; substrate always formate a hydrogen-bond with Gly392.3 Trametes versicolor laccase is a good candidate for design. We establish F162 A mutant, D206 A mutant, F265 A mutation, F332 A mutation, P391 A mutation, P391 L mutation and F162A/F332 A mutation structural models. We believe that F162 A mutant, F332 A mutation, P391 A mutation and F162A/F332 A mutation are promising directions based on the interaction between mutation laccases and VX. We obtain the P391 A mutation laccase which has good effect by theory predicts, and will test its activity in further study.