KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

Chemoenzymatic Synthesis of a New Germacrene Derivative Named Germacrene F

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6-C7 to C7-C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name “germacrene F”. Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature-dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror-symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring

In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators

Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regu lated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM170 s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modula tors as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibi tion mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity

Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

Cellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability

Lipase-Mediated Conversion of Protecting Group Silyl Ethers: An Unspecific Side Reaction

Silyl ether protecting groups are important tools in organic synthesis, ensuring selective reactions of hydroxyl functional groups. Enantiospecific formation or cleavage could simultaneously enable the resolution of racemic mixtures and thus significantly increase the efficiency of complex synthetic pathways. Based on reports that lipases, which today are already particularly important tools in chemical synthesis, can catalyze the enantiospecific turnover of trimethylsilanol (TMS)-protected alcohols, the goal of this study was to determine the conditions under which such a catalysis occurs. Through detailed experimental and mechanistic investigation, we demonstrated that although lipases mediate the turnover of TMS-protected alcohols, this occurs independently of the known catalytic triad, as this is unable to stabilize a tetrahedral intermediate. The reaction is essentially non-specific and therefore most likely completely independent of the active site. This rules out lipases as catalysts for the resolution of racemic mixtures of alcohols through protection or deprotection with silyl groups

How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

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. KGa

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation

Disulfide-induced self-assembled targets:A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol-modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3' and 5' terminals of targets leads to the self-assembly of dsDNAs into the sulfur-rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 x 10(-9) mol. L-1 over a linear ranged from 20 x 10(-9) to 10 x 10(-7) mol. L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications

Rational reprogramming of the sesquiterpene synthase BcBOT2 yields new terpenes with presilphiperfolane skeleton

Computer-aided rational design of the substrate binding pocket of sesquiterpene synthases BcBOT2 from Botrytis cinerea yielded FPP cyclization products with presilphiperfolane backbone other than the naturally formed sesquiterpene presilphiperfolan-8β-ol. Particularly, amino acids W118 and F138 were found to strongly control the stability and conformation of key cationic intermediates. The W118Q variant forms only presilphiperfolan-9β-ol, whereas the exchange of amino acids at position 138, such as F138V, has a fundamental effect on the course of the cationic cascade. Here, the 1,3-hydride shift en route to presilphiperfolan-8β-ol is suppressed and substituted by a so far unknown 1,2-hydride shift that leads to presilphiperfol-1-ene and presilphiperfolan-1α-ol along with β-caryophyllene and the so far unknown caryophyllene-8β-ol

A hydroquinone-specific screening system for directed P450 evolution

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). © 2018, The Author(s)