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- ItemLess Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance(Weinheim : Wiley-VCH, 2021) Cui, Haiyang; Eltoukhy, Lobna; Zhang, Lingling; Markel, Ulrich; Jaeger, Karl-Erich; Davari, Mehdi D.; Schwaneberg, UlrichBiocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other α/β-hydrolases. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
- ItemHow to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study(Weinheim : Wiley-VCH, 2020) Cui, Haiyang; Stadtmüller, Tom H.J.; Jiang, Qianjia; Jaeger, Karl-Erich; Schwaneberg, Ulrich; Davari, Mehdi D.Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4-dioxane, dimethyl sulfoxide, 2,2,2-trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H-bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with “BSLA-SSM” variant library (3439 variants; all natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
- ItemComputer-Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns(Weinheim : Wiley-VCH, 2020) Cui, Haiyang; Cao, Hao; Cai, Haiying; Jaeger, Karl-Erich; Davari, Mehdi D.; Schwaneberg, UlrichA main remaining challenge in protein engineering 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. This limits researchers in exploiting 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 (ΔΔGfold). The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of Bacillus subtilis lipase A. The finally obtained variant F17S/V54K/D64N/D91E had a 2.7-fold improved specific activity in 18.3 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). In essence, the deducted CompassR rule allows recombination of beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
- ItemEngineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design(Weinheim : Wiley-VCH, 2020) Zhang, Lingling; Cui, Haiyang; Dhoke, Gaurao V.; Zou, Zhi; Sauer, Daniel F.; Davari, Mehdi D.; Schwaneberg, UlrichCopper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at −250 mV (up to 6.13 mA cm−2). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.