Molecular docking of substrates is more challenging compared to inhibitors as the reaction mechanism has to be considered. This becomes more pronounced for zinc-dependent enzymes since the coordination state of the catalytic zinc ion is of greater importance. In order to develop a predictive substrate docking protocol, we have performed molecular docking studies of diketone substrates using the catalytic state of carbonyl reductase 2 from Candida parapsilosis CPCR2. Different docking protocols using two docking methods, AutoDock Vina and AutoDock4.2, with two different sets of atomic charges, AM1-BCC and HF-RESP, for catalytic zinc environment and substrates as well as two sets of vdW parameters for zinc ion were examined. We have selected the catalytic binding pose of each substrate by applying mechanism based distance criteria. To compare the performance of the docking protocols, the correlation plots for the binding energies of these catalytic poses were obtained against experimental Vmax values of the 11 diketone substrates for CPCR2. The best correlation of 0.73 was achieved with AutoDock4.2 while treating catalytic zinc ion in optimized non-bonded NBopt state with ?1.01 charge on the zinc ion, compared to 0.36 in non-bonded - ?2.00 charge on the zinc ion - state. These results indicate the importance of catalytic constraints and charge parameterization of catalytic zinc environment for the prediction of substrate activity in zinc-dependent enzymes by molecular docking. The developed predictive docking protocol described here is in principle generally applicable for the efficient in silico substrate spectra characterization of zinc-dependent ADH.Wiley-VCH
Rhodium-Complex-Linked Hybrid Biocatalyst:
Stereo-Controlled Phenylacetylene Polymerization within an Engineered Protein Cavity The incorporation of a Rh complex with a maleimide moiety into the cavity of the nitrobindin b-barrel scaffold by a covalent linkage at the 96-position (Cys) provides a hybrid biocatalyst that promotes the polymerization of phenylacetylene.
The appropriate structural optimization of the cavity by mutagenesis enhances the stereoselectivity of the polymer with a trans content of 82% at 258C and pH 8.0. The X-ray crystal structure of one of the hybrid biocatalysts at a resolution of 2.0 reveals that the Rh complex is located in the b-barrel cavity without any perturbation to the total protein structure.
Crystal structure analysis and molecular modeling support the fact that the stereoselectivity is enhanced by the effective control of monomer access to the Rh complex within the limited space of the protein cavity.
Shehzad, A., Panneerselvam, S., Linow, M., Bocola, M., Roccatano, D., Mueller-Dieckmann, J., Wilmanns, M., and Schwaneberg, U. (2013). P450 BM3 crystal structures reveal the role of the charged surface residue Lys/Arg184 in inversion of enantioselective styrene epoxidation. Chem. Commun., 2013, 49, 4694-4696.Bio VI
Overview of the ten generated NB variants. a) The amino acid residues in the vicinity of the Rh complex within NB(Q96C)-Rh. Two calculated stable conformations of the Rh complex are shown. The Rh atom and its ligand are represented by pink and purple sticks, respectively. Met75 and Met148 were replaced by Leu in all variants. b) List of re-engineered NB variants for stereoselective PPA synthesis.Bio VI
Study the enantioselective styrene epoxidation in P450 BM3 and to explore how a single charged surface residue, Lys/Arg184, inverts the enantioselectivity in P450 BM3 variants 5F5K - F87A, T235A, A184K, and 5F5R - F87A, T235A, A184R, as compared to the parent 5F5 - F87A, T235A, variant.
Substitution of Lys/Arg for Ala in 5F5K StB and 5F5R StB structures establishes a salt-bridge between Lys/Arg184 - F-helix, and Asp80 - B’-helix.
The salt-bridge relays structural changes from the surface of P450 BM3 muteins to the active site and stabilizes 5F5K StB and 5F5R StB structures in WTSB forms - reduced I-helix kink angle.
Due to kinked I-helix, Ala264 covers completely the heme pyrrole ring C, and provides sufficient steric hindrance to prevent exposure of the pro-S face of styrene to the heme-iron which, as a result, promotes the formation of R-styrene epoxide as the preferred enantiomer in the 5F5 variant.
Dhoke, G. V., Davari, M. D., Schwaneberg, U., Bocola, M. 2015. QM/MM Calculations Revealing the Resting and Catalytic States in Zinc-Dependent Medium-Chain Dehydrogenases/Reductases. ACS Catalysis, 5, 3207-3215.
Loderer, C., Dhoke, G. V., Davari, M. D., Kroutil, W., Schwaneberg, U., Bocola, M., Ansorge-Schumacher, M. B. 2015. Investigation of structural determinants for the substrate specificity in the zinc-dependent alcohol dehydrogenase CPCR2 from Candida parapsilosis. ChemBioChem, 16, 1512-1519.
Dhoke, G. V., Loderer, C., Davari, M. D., Ansorge-Schumacher, M., Schwaneberg, U., Bocola, M. 2015. Activity prediction of substrates in NADH-dependent carbonyl reductase by docking requires catalytic constraints and charge parameterization of catalytic zinc environment. J. Comput. Aided Mol. Des., 29, 1057-1069