Computational protocol: A new target region for changing the substrate specificity of amine transaminases

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Protocol publication

[…] The initial phasing of Ab-R-ATA diffraction data was conducted by molecular replacement using MOLREP in the CCP4 and with a single molecule of branched-chain amino acid aminotransferase as the search model (PDB 1IYE). The initial solution with one molecule in the asymmetric unit had starting Rwork and Rfree values in excess of 50%, which only dropped slightly, respectively, after refinement with Refmac5. The model was therefore improved using Buccaneer and ARP/wARP, and these software successfully built most of the structure. The structure was then refined using Refmac5 and Phenix to obtain a model containing water with Rwork and Rfree values of 18.0% and 22.7%, respectively. The structure refinement was completed in Coot and Refmac5. The crystal structures of ATA-117-Rd11 and the G136F mutant were solved by molecular replacement using the structure of the Ab-R-ATA as a search model. The structures were manually rebuilt using Coot and refined using Refmac5. [...] The model of the wild-type Ab-R-ATA complexed with PMP-pyruvate quinonoid intermediate was generated by using software MOE2014.09 (Chemical Computing Group, Montreal, Canada) from the atomic coordinates of the wild-type Ab-R-ATA complexed with PLP in the internal aldimine form. The Schiff linkage of the internal aldimine was removed and the quinonoid intermediate was built by adding atoms to the PLP residue. The quinonoid and the side chain of Lys188 were energy-minimized using AMBER12:EHT force field with distance-dependent dielectric electrostatics, while the other atoms are fixed. The homodimer of Ab-R-ATA was generated by using crystallographic symmetry operation.Preparation of system was also carried out with MOE2014.09. The missing hydrogen atoms were generated by Protonate 3D function and energy-minimized using AMBER12:EHT force field with distance-dependent dielectric electrostatics. Sodium ions for neutralization and explicit water molecules were placed around the protein and the cubic periodic boundary was set at least 4-Å apart from the protein surface. The ions and solvent molecules were energy-minimized and applied to 450-ps dynamics simulation in order to be fit to the protein surface appropriately. All atoms in the system were then energy-minimized. Gradually decreased tether weight was applied to all non-hydrogen atoms during this final minimization steps.Molecular dynamics simulation was carried out using software NAMD 2.10 using the input file generated by MOE2014.09. The first 1-ns simulation, which was not used for trajectory analysis, was performed with the gradually decreasing positional restrains for non-hydrogen atoms. Then, molecular dynamics simulation without restrains were performed and used for trajectory analysis. All the dynamics simulations using NAMD were carried out with the time step of 1 fs in NVT ensemble at pressure of 101 kPa and at temperature of 300 K without any bonding constraints. Langevin method was used to control the constant temperature and pressure. The short range interactions were cut off at 10 Å for van der Waals and electrostatic interactions and long-range electrostatic interactions were calculated using the particle-mesh Ewald method. Obtained trajectory was analyzed by using software VMD. […]

Pipeline specifications

Software tools Molrep, CCP4, REFMAC5, Buccaneer, ARP/wARP, PHENIX, Coot, NAMD, VMD
Application Protein structure analysis
Diseases Diabetes Mellitus
Chemicals Amines