Theoretical And Computational Researches On Carboxyltransferase Domain Of Acetyl Coenzyme A Carboxylase

Acetyl coenzyme A carboxylase (ACCase) is an important enzyme associated with fatty acid metabolism. In plants, the poaceae ACCase, which includes many species of weed grass, is different with the one from non-poaceae in structural organization, and thus is considered as an effective herbicide target in the new herbicide development. The class of Aryloxyphenoxypropionic acid herbicide is a wildly used commercial herbicide, which selectively inhibited the ACCase carboxyltransferase (CT) domain from poaceae. It has a pair of enantiomers, and the inhibitive activity of (R)-enantiomer is two orders of magnitude higher than the (S)-haloxyfop, the molecular mechanism is arguable. Recently, as the abuse use of this kind of herbicide, more and more resistant poaceae plants have emerged. The most resistant casees are observed to be caused by the single residue mutatation in ACCase CT. Among them, an Ile/Leu mutation is of especial interest. It is because this mutation has widely occurred in many poaceae plants and the residues have subtle difference before and after mutation. Facing the more and more challenging resistant situation, to develop new efficient herbicides targeting the ACCase is urgent. During this process, it is of fundament importance to understand interaction mechanism between CT and APPs, such as why the CT is enantioselectively inhibited by the (R)-enantiomers and how this Ile/Leu single mutation causes the resistance. In this dissertation, we try to solve these questions by theoretical and computational methods, and the details are as following:At first, in order to solve the time-costing computing trouble caused by the big magnitude of CT active dimmer. We optimized the CT dimmer and molecular dynamics parameters by combining the molecular dynamics simulation and referring literature to develop an efficient truncated CT dimmer and optimized parameters. Results show these optimizations can largely reduced the computing time and not effect the computing results and precision. In the following researches, these model and optimized parameters are accepted.Next, we constructed the structures of CT from black grass (Alopecurus myosurodies) and its complex with (R)- or (S)-haloxyfop by homology modeling, and calculated binding free energy and their components for both (R)- and (S)-haloxyfop binding by molecular dynamics and MM-PBSA method, followed by a detail analysis of the various contributions to their difference. Results show that for both enantiomers binding, the intermolecualr van der Waals interaction energy is the driving force, non-polar salvation energy has little contribution to the binding, the total electrostatic energy between the molecules is adverse, and the conformational change energy, which originate from the intramolecular interaction is also adverse. As regards to the binding free energy difference, it is suggested that the intermolecualr van der Waals interaction energy difference is the determinant of the total binding free energy difference, the conformational change energy difference only has a minor contribution, and the intermolecular total electrostatic interaction difference and non-polar solvation difference have little contributions. All these results are consistent with the fact that the CT binding site is a hydrophobic pocket, and the most residues nearby are hydrophobic. Further research also show the individual residues have almost same contributions for (R)- and (S)-haloxyfop binding to the CT, suggesting the enantioselectivity of CT to haloxyfop is the sum of individual residue contribution. In the end, with regard to the proposal that the enantioselectivity arise from the intermolecular interaction, we examine the deduction process again, and found there is an ignore in the deduction. If we consider it, the results turn to support the proposal that the intermolecular interaction difference determined the enantioselectivity, which further confirm our results.In the next, we also constructed the structures of wild and mutated CT complex with (R)-haloxyfop for black grass and Lolium rigidum, and calculated their binding free energies to the haloxyfop and each contribution terms by molecular dynamics and MM-PBSA method, followed by a detail analysis of the various contributions to this binding free energy difference. Results indicate that the situation is similar with the calculations for the enantiomers. For all haloxyfp-binding of wild-type and mutated CT from black grass or Lolium rigidum, the intermolecular van der Waals interaction energy is major driving force, the non-polar solvation energy has little contribution, and the intermolecular total electrostatic interaction energy is adverse. For the haloxyfop binding difference between the wild-type and mutated CT, the intermolecular van der Waals interaction energy difference is the major determinant and total electrostatic interaction energy difference and non-polar solvation energy difference has little effect. Since both in black grass or Lolium rigidum, similar results are observed. It is suggested that the APPs-resitance caused by Ile/Leu mutation in different species of poaceae may caused by the same mechanism. Next, the complex structures of Lolium rigidum wild-type and mutated CT with haloxyfop are chosen to analyze structure difference. We found in the complex structure of mutated CT and haloxyfop there is a steric hindrance between the sidechain of Leu and the methyl group of chiral carbon atom. However, in the complex structure of wild-type CT and haloxyfop, this steric hindrance is not found. It is consist with our above energy calculation, suggesting the van der Waals interaction energy difference caused by Ile/Leu mutation is the major determinant which arise the resistance.In all, our woriking provide an important foundation and a good clue for design of new herbicides against the ACCase CT.