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Title: Modelling of fatty acid amide hydrolase and aristolochene synthase from Aspergillus terreus
Author: Sirirak, Jitnapa
ISNI:       0000 0004 2717 238X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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Fatty acid amide hydrolase (FAAH) is a promising drug target for many nervous system disorder therapies. Understanding its mechanism could help in designing new inhibitors. In this work, the acylation reaction mechanisms of oleamide (OLE) and oleoylmethylester (OME) hydrolysis by FAAH were investigated by computational simulation methods, building on previous work. Umbrella sampling molecular dynamics simulations were initially carried out at the PM3/CHARMM27 level of theory, to calculate free energy profiles for the reactions. Adiabatic mapping at high levels of QMlMM theory was used to generate potential energy profiles, which were then refined by nudged elastic band methods. The results from these three methods showed similar reaction mechanisms, which were also similar to the experimental proposed mechanism. One water molecule was found to influence the OLE reaction pathway in the adiabatic mapping profiles. Compared with the experimental values, the PM3/CHARMM27 method significantly overestimated the activation barriers for OLE and OME (by -20 kcal/mol). Higher levels of QM/MM theory gave activation barriers of 14 and 20 kcal/mol for OLE and OME, respectively, (calculated at the SCS-MP2/aug-cc-pvdz QM/MM level), which were comparable to the experimental values. Compared to SCS-MP2 and MP2 methods, although both B3L yP and BH&HL YP give small errors in the estimated energy of tetrahedral intermediate relative to substrate, these two DFT methods provide the good structures with reliable energies for these FAAH reactions, determined in agreement with the experimental findings. In the second part of the work, molecular dynamics simulations were used to study the conformational change of aristolochene synthase from Aspergillus terreus (ATAS) associated with the catalytic cycle, in order to obtain insight into the AT AS function. This could help in the generating new biosynthesis products. The simulation results of 16 systems of' enzyme-substrate, enzyme-substrate-Mg'", and enzyme-Mg'" -pyrophosphate anion (PPi) complexes with varied position and number of Mg2+ (30 ns each) show that PPj and only two Mg2+ ions. at positions A and B are required to keep the closed conformation of AT AS, and the third Mg2+ ion (Mg2+ (C)) is required to keep the active site template of the closed conformation of ATAS for catalysis. The preferred conformation of each system observed here agree well with the ATAS crystal structures, and support the conformational changes in the proposed metal binding sequence for catalysis by Shishova et al.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available