CompassR: A new rule for recombination of beneficial substitutions in directed evolutionCopyright: © BioVI
Haiyang Cui, Hao Cao, Haiying Cai, Karl Erich Jaeger, Mehdi D. Davari, Ulrich Schwaneberg, Computer‐assisted Recombination (CompassR) teaches us how to recombine beneficial substitutions from directed evolution campaigns, Chemistry A European Journal, 2019, https://doi.org/10.1002/chem.201903994
The Computer-assisted Recombination – CompassR – strategy is a selection filter for experimentalists to recombine beneficial substitutions in order to gradually improve enzyme performance and maximize improvements through recombination.
Directed evolution has matured into a powerful method for improving enzymes in catalysis and medical applications as documented by the Nobel Prize in chemistry in 2018. A main remaining challenge is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. The latter limits researchers to harness the benefits from beneficial substitutions that have been identified in directed evolution campaigns and to exploit nature’s potential in generating better enzymes. The Computer-assisted Recombination – CompassR – strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding. The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of the Bacillus subtilis lipase A. Analysis of their relative free energy of folding was used to deduct the CompassR rule that substitutions with a relative free energy of folding below +0.36 kcal/mol can be efficiently recombined, variants with a free energy of folding between +0.36 and +7.52 kcal/mol were active and inactive indicating unpredictable behavior, folding energies over +7.52 kcal/mol mostly yielded inactive variants. The finally obtained recombinant with four amino acid substitutions had a 2.7-fold improved specific activity in 18.3 % ionic liquid [BMIM][Cl]. Notably, the BSLA variants with 3 and 4 substitutions were better performing than the variants with 2 substitutions. In essence, the deducted CompassR allows to recombine beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner.
This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187). Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship.
Molecular understanding of enzyme and ionic liquid interactions provide a general protein engineering strategy for their sustainable biocatalytic applicationsCopyright: © BIO VI
Pramanik S.†, Dhoke G. V.†, Jaeger KE., Schwaneberg U., Davari MD. ACS Sustainable Chem. Eng., 2019, https://doi.org/10.1021/acssuschemeng.9b00752 †shared co-authorship
Copyright: © ACS Sustainable Chem. Eng.
A general protein engineering strategy was suggested through molecular understanding of enzyme and ionic liquid interactions based on computational study.
In this publication, interactions between Bacillus subtilis lipase A (BSLA) and four imidazolium-based ionic liquids were studied using molecular dynamic simulations. This study provides first insight that ionic liquids co-solvents do not alter the overall and local BSLA conformation. The effect on the reduction of activity was attributed to surface interactions of the ionic liquid cations, which removed essential water molecules from the BSLA surface.Furthermore, the comparison of simulation results with experimental full site-saturation mutagenesis BSLA libraries confirmed that most of beneficial positions for resistance improvement are located at the ionic liquid cations binding regions. Taken together, these findings suggest that surface charge engineering might be a general protein engineering strategy to improve BSLA resistance in ionic liquids and is most likely applicable to other lipases and α/β-hydrolases.
This work was realized in the division of Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187).
Enzyme-polyelectrolyte complexes boost the catalytic performance of enzymesCopyright: © Bio VI
Martin J. Thiele, Mehdi D. Davari, Melanie König, Isabell Hofmann, Niklas O. Junker, Tayebeh Mirzaei Garakani, Ljubica Vojcic, Jörg Fitter, Ulrich Schwaneberg, ACS Catalysis, 2018, DOI: 10.1021/acscatal.8b02935
In the present publication, a molecular understanding was reported on polyelectrolyte-enzyme complexes which boost enzymatic activity in multi-component systems.
In this publication, a molecular understanding of the boosting effect of polyacrylic acid -PAA-) and poly-L-γ-glutamic acid -γ-PGA- for a nonspecific subtilisin protease was generated through biophysical characterization, e.g. fluorescence correlation and circular dichroism spectroscopies, isothermal titration calorimetry, molecular dynamics simulations and protease reengineering, e.g. site-saturation mutagenesis. This study revealed that enthalpically driven interactions via key amino acid residues close to the protease Ca2+ binding sites cause the boosting effect in protease activity. On the molecular level electrostatic interactions result in the formation of protease-polyelectrolyte complexes. Site-saturation mutagenesis on 6 positions yielded an increased proteolytic performance against a complex protein mixture -trademark CO3; up to ~300% and ~70%- in the presence of PAA and γ-PGA. Being able to fine-tune interactions between proteins and negatively charged polymers through integrative use of computational design, protein reengineering and biophysical characterization proved to be an efficient workflow to improve protease performance.
This research was partially funded by Henkel AG & Co. KGaA, Düsseldorf, Germany, as part of the Henkel Innovation Campus for Advanced Sustainable Technologies -HICAST- project. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169.
Enzyme-compatible dynamic nanoreactors from electrostatically bridged like-charged surfactants and polyelectrolytesCopyright: © Bio VI
Martin J. Thiele, Mehdi D. Davari, Isabell Hofmann, Melanie König, Carlos G. Lopez, Ljubica Vojcic, Walter Richtering, Ulrich Schwaneberg and Larisa A. Tsarkova, Angewandte Chemie, 2018, DOI: 10.1002/anie.201805021
In the present publication, a discovery of nanoreactors from electrostatically bridged like-charged surfactants and polyelectrolytes was reported. Incorporation of a protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance due to the improved solubilization accessibility for the protease through the interaction with the nanoreactors.
An unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid with like-charged surfactants is reported. Amphiphilic polymer-surfactant complexes with high interfacial activity and a solubilization capacity exceeding that of conventional micelles are formed by bridging with Ca2+ ions. Incorporation of a non-specific protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance due to improved solubilization of poorly water-soluble immobilized proteins. Competitive interfacial and intermolecular interactions on different time- and length-scales have been resolved using colorimetric analysis, dynamic tensiometry, light scattering, and molecular dynamic simulations. The discovered bridging association mechanism suggests reengineering of surfactant/polymer/enzyme formulations of modern detergents and opens new opportunities in advancing labile delivery systems.
Image: Scheme of an unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid (PAA) with like-charged surfactant SLES. Amphiphilic polymer-surfactant complexes with high interfacial activity and solubilization capacity are formed by bridging with Ca2+ ions. Incorporation of a protease into such dynamic nanoreactors results in an enhanced cleaning performance because of the improved solubilization of poorly water-soluble immobilized proteins.
This research was partially funded by Henkel AG & Co. KGaA, Düsseldorf, Germany, as part of the Henkel Innovation Campus for Advanced Sustainable Technologies (HICAST). Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169. L.A.T. acknowledges financial support by the Russian Foundation for Basic Research (RFBR) according to the research project No 18-53-76005.
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.Copyright: © 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.Copyright: © 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.Copyright: © 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