03 Fakultät Chemie
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Item Open Access The triple variant K170D/N174L/D239A compensates the destabilizing effect of variant K170D/N174L in β-hydroxyacid dehydrogenase (βHAD) from Arabidopsis thaliana(2020) Schelle, Luca S.; Stockinger, Peter; Pleiss, Jürgen; Nestl, Bettina M.Chiral amines are essential building blocks in biologically active compounds, fine chemicals, agrochemicals and pharmaceuticals. In the last ten years, various enzymes were identified as new biocatalysts for chiral amine synthesis. Promising enzymes for the synthesis of primary, secondary, and tertiary amines are NADPH-dependent imine reductases (IREDs). Bioinformatics analysis revealed that IREDs are closely related to β-hydroxyacid dehydrogenases (βHADs). In recent work, we engineered the βHAD from Arabidopsis thaliana (βHAD_At) into imine-reducing enzymes by a single amino acid exchange. The exchange of the proton-donor described lysine (K170) in βHAD_At by aspartic acid, the most common amino acid at this position in R-selective IREDs, led to a 12-fold increase in activity for the model substrate 2-methylpyrroline. At the same time, the activity for the natural substrate glyoxylic acid is reduced 885-fold, resulting in a total of 8200-fold change in catalytic activity through the exchange of an amino acid. At the same time, highly decreased soluble expression has been observed by exchanging asparagine at position 174 (N174) with leucine. We thus hypothesized, that the aspartic acid residue (D239) in near proximity to N174 will stabilize the underlying α-helix. Consequently, replacement of D239 with alanine should result in soluble expression of variants containing the N174 mutations. We generated variants K170D/D239A, as well as, and tested them on imine reduction of test substrates 2-methylpyrroline, 3,4-dihydroisoquinoline and 6-phenyl-2,3,4,5-tetrahydropyridine. Due to loss of essential cofactor and precipitation of purified proteins during purification procedure, activities of variants were determined using cell lysates. Notably, variants N174L/D239A and K170D/N174L/D239A demonstrated soluble expression and imine-reducing activities of up to 98 mU per mg of variant.Item Open Access Visual analysis of large‐scale protein‐ligand interaction data(2021) Schatz, Karsten; Franco‐Moreno, Juan José; Schäfer, Marco; Rose, Alexander S.; Ferrario, Valerio; Pleiss, Jürgen; Vázquez, Pere‐Pau; Ertl, Thomas; Krone, MichaelWhen studying protein‐ligand interactions, many different factors can influence the behaviour of the protein as well as the ligands. Molecular visualisation tools typically concentrate on the movement of single ligand molecules; however, viewing only one molecule can merely provide a hint of the overall behaviour of the system. To tackle this issue, we do not focus on the visualisation of the local actions of individual ligand molecules but on the influence of a protein and their overall movement. Since the simulations required to study these problems can have millions of time steps, our presented system decouples visualisation and data preprocessing: our preprocessing pipeline aggregates the movement of ligand molecules relative to a receptor protein. For data analysis, we present a web‐based visualisation application that combines multiple linked 2D and 3D views that display the previously calculated data The central view, a novel enhanced sequence diagram that shows the calculated values, is linked to a traditional surface visualisation of the protein. This results in an interactive visualisation that is independent of the size of the underlying data, since the memory footprint of the aggregated data for visualisation is constant and very low, even if the raw input consisted of several terabytes.Item Open Access Fluent integration of laboratory data into biocatalytic process simulation using EnzymeML, DWSIM, and ontologies(2024) Behr, Alexander S.; Surkamp, Julia; Abbaspour, Elnaz; Häußler, Max; Lütz, Stephan; Pleiss, Jürgen; Kockmann, Norbert; Rosenthal, KatrinThe importance of biocatalysis for ecologically sustainable syntheses in the chemical industry and for applications in everyday life is increasing. To design efficient applications, it is important to know the related enzyme kinetics; however, the measurement is laborious and error-prone. Flow reactors are suitable for rapid reaction parameter screening; here, a novel workflow is proposed including digital image processing (DIP) for the quantification of product concentrations, and the use of structured data acquisition with EnzymeML spreadsheets combined with ontology-based semantic information, leading to rapid and smooth data integration into a simulation tool for kinetics evaluation. One of the major findings is that a flexibly adaptive ontology is essential for FAIR (findability, accessibility, interoperability, reusability) data handling. Further, Python interfaces enable consistent data transfer.Item Open Access EnzymeML : a data exchange format for biocatalysis and enzymology(2021) Range, Jan; Halupczok, Colin; Lohmann, Jens; Swainston, Neil; Kettner, Carsten; Bergmann, Frank T.; Weidemann, Andreas; Wittig, Ulrike; Schnell, Santiago; Pleiss, JürgenEnzymeML is an XML‐based data exchange format that supports the comprehensive documentation of enzymatic data by describing reaction conditions, time courses of substrate and product concentrations, the kinetic model, and the estimated kinetic constants. EnzymeML is based on the Systems Biology Markup Language, which was extended by implementing the STRENDA Guidelines. An EnzymeML document serves as a container to transfer data between experimental platforms, modeling tools, and databases. EnzymeML supports the scientific community by introducing a standardized data exchange format to make enzymatic data findable, accessible, interoperable, and reusable according to the FAIR data principles. An application programming interface in Python supports the integration of software tools for data acquisition, data analysis, and publication. The feasibility of a seamless data flow using EnzymeML is demonstrated by creating an EnzymeML document from a structured spreadsheet or from a STRENDA DB database entry, by kinetic modeling using the modeling platform COPASI, and by uploading to the enzymatic reaction kinetics database SABIO‐RK.Item Open Access Plastics degradation by hydrolytic enzymes : the Plastics-Active Enzymes Database - PAZy(2022) Buchholz, Patrick C. F.; Feuerriegel, Golo; Zhang, Hongli; Perez‐Garcia, Pablo; Nover, Lena‐Luisa; Chow, Jennifer; Streit, Wolfgang R.; Pleiss, JürgenPetroleum‐based plastics are durable and accumulate in all ecological niches. Knowledge on enzymatic degradation is sparse. Today, less than 50 verified plastics‐active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Furthermore, very few polyamide (PA) oligomer active enzymes are known. In this article, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their published activity data were collected and integrated into a comprehensive open access database. The Plastics‐Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified enzymes that act on synthetic fossil fuel‐based polymers. Almost 3000 homologs of PET‐active enzymes were identified by profile hidden Markov models. Over 2000 homologs of PUR‐active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET‐ and PUR‐active enzymes were derived.Item Open Access Expansin Engineering Database : a navigation and classification tool for expansins and homologues(2020) Lohoff, Caroline; Buchholz, Patrick C. F.; Le Roes‐Hill, Marilize; Pleiss, JürgenExpansins have the remarkable ability to loosen plant cell walls and cellulose material without showing catalytic activity and therefore have potential applications in biomass degradation. To support the study of sequence‐structure‐function relationships and the search for novel expansins, the Expansin Engineering Database (ExED, https://exed.biocatnet.de) collected sequence and structure data on expansins from Bacteria, Fungi, and Viridiplantae, and expansin‐like homologues such as carbohydrate binding modules, glycoside hydrolases, loosenins, swollenins, cerato‐platanins, and EXPNs. Based on global sequence alignment and protein sequence network analysis, the sequences are highly diverse. However, many similarities were found between the expansin domains. Newly created profile hidden Markov models of the two expansin domains enable standard numbering schemes, comprehensive conservation analyses, and genome annotation. Conserved key amino acids in the expansin domains were identified, a refined classification of expansins and carbohydrate binding modules was proposed, and new sequence motifs facilitate the search of novel candidate genes and the engineering of expansins.Item Open Access Meta-analysis of viscosity of aqueous deep eutectic solvents and their components(2020) Gygli, Gudrun; Xu, Xinmeng; Pleiss, JürgenDeep eutectic solvents (DES) formed by quaternary ammonium salts and hydrogen bond donors are a promising green alternative to organic solvents. Their high viscosity at ambient temperatures can limit biocatalytic applications and therefore requires fine-tuning by adjusting water content and temperature. Here, we performed a meta-analysis of the impact of water content and temperature on the viscosities of four deep eutectic solvents (glyceline, reline, N,N-diethylethanol ammonium chloride-glycerol, N,N-diethylethanol ammonium chloride-ethylene glycol), their components (choline chloride, urea, glycerol, ethylene glycol), methanol, and pure water. We analyzed the viscosity data by an automated workflow, using Arrhenius and Vogel-Fulcher-Tammann-Hesse models. The consistency and completeness of experimental data and metadata was used as an essential criterion of data quality. We found that viscosities were reported for different temperature ranges, half the time without specifying a method of desiccation, and in almost half of the reports without specifying experimental errors. We found that the viscosity of the pure components varied widely, but that all aqueous mixtures (except for reline) have similar excess activation energy of viscous flow Eexcessη= 3-5 kJ/mol, whereas reline had a negative excess activation energy (Eexcessη= - 19 kJ/mol). The data and workflows used are accessible at https://doi.org/10.15490/FAIRDOMHUB.1.STUDY.767.1.Item Open Access Inverting the stereoselectivity of an NADH‐dependent imine‐reductase variant(2021) Stockinger, Peter; Borlinghaus, Niels; Sharma, Mahima; Aberle, Benjamin; Grogan, Gideon; Pleiss, Jürgen; Nestl, Bettina M.Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)‐selective IRED from Myxococcus stipitatus (NADH‐IRED‐Ms) yielding a NADH‐dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)‐selectivity in asymmetric reductions has yet been reported. Herein, we applied semi‐rational enzyme engineering to switch the selectivity of NADH‐IRED‐Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2‐methylpyrroline compared to the wild‐type and afforded the (S)‐amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal‐structures of the NADPH‐dependent (R)‐IRED‐Ms wild‐type enzyme and the NADH‐dependent NADH‐IRED‐Ms variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.Item Open Access FAIR and scalable management of small‐angle X‐ray scattering data(2023) Giess, Torsten; Itzigehl, Selina; Range, Jan; Schömig, Richard; Bruckner, Johanna R.; Pleiss, JürgenA modular research data management toolbox based on the programming language Python, the widely used computing platform Jupyter Notebook, the standardized data exchange format for analytical data (AnIML) and the generic repository Dataverse has been established and applied to analyze small‐angle X‐ray scattering (SAXS) data according to the FAIR data principles (findable, accessible, interoperable and reusable). The SAS‐tools library is a community‐driven effort to develop tools for data acquisition, analysis, visualization and publishing of SAXS data. Metadata from the experiment and the results of data analysis are stored as an AnIML document using the novel Python‐native pyAnIML API. The AnIML document, measured raw data and plots resulting from the analysis are combined into an archive in OMEX format and uploaded to Dataverse using the novel easyDataverse API, which makes each data set accessible via a unique DOI and searchable via a structured metadata block. SAS‐tools is applied to study the effects of alkyl chain length and counterions on the phase diagrams of alkyltrimethylammonium surfactants in order to demonstrate the feasibility and usefulness of a scalable data management workflow for experiments in physical chemistry.Item Open Access The triple variant K170D/N174L/D239A compensates the destabilizing effect of variant K170D/N174L in β-hydroxyacid dehydrogenase (βHAD) from Arabidopsis thaliana(2020) Schelle, Luca S.; Stockinger, Peter; Pleiss, Jürgen; Nestl, Bettina M.Chiral amines are essential building blocks in biologically active compounds, fine chemicals, agrochemicals and pharmaceuticals. In the last ten years, various enzymes were identified as new biocatalysts for chiral amine synthesis. Promising enzymes for the synthesis of primary, secondary, and tertiary amines are NADPH-dependent imine reductases (IREDs). Bioinformatics analysis revealed that IREDs are closely related to β-hydroxyacid dehydrogenases (βHADs). In recent work, we engineered the βHAD from Arabidopsis thaliana (βHAD_At) into imine-reducing enzymes by a single amino acid exchange.[7] The exchange of the proton-donor described lysine (K170) in βHAD_At by aspartic acid, the most common amino acid at this position in R-selective IREDs, led to a 12-fold increase in activity for the model substrate 2-methylpyrroline. At the same time, the activity for the natural substrate glyoxylic acid is reduced 885-fold, resulting in a total of 8200-fold change in catalytic activity through the exchange of an amino acid. At the same time, highly decreased soluble expression has been observed by exchanging asparagine at position 174 (N174) with leucine. We thus hypothesized, that the aspartic acid residue (D239) in near proximity to N174 will stabilize the underlying α-helix. Consequently, replacement of D239 with alanine should result in soluble expression of variants containing the N174 mutations. We generated variants K170D/D239A, N174L/D239A and K170D/N174L/D239A, and tested them on imine reduction of test substrates 2-methylpyrroline, 3,4-dihydroisoquinoline and 6-phenyl-2,3,4,5-tetrahydropyridine. Due to loss of essential cofactor and precipitation of purified proteins during purification procedure, activities of variants were determined using cell lysates. Notably, variants N174L/D239A and K170D/N174L/D239A demonstrated soluble expression and imine-reducing activities of up to 98 mU per mg of variant.