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  1. Article ; Online: Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

    Hermann, Enikö / Rodrigues, Carolina F / Martins, Lígia O / Peterbauer, Clemens / Oostenbrink, Chris

    Chembiochem : a European journal of chemical biology

    2024  Volume 25, Issue 9, Page(s) e202300872

    Abstract: Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many ... ...

    Abstract Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.
    MeSH term(s) Glutamic Acid/chemistry ; Glutamic Acid/metabolism ; Coloring Agents/chemistry ; Coloring Agents/metabolism ; Bacillus subtilis/enzymology ; Peroxidases/chemistry ; Peroxidases/metabolism ; Peroxidases/genetics ; Molecular Dynamics Simulation ; Protein Engineering ; Mutagenesis, Site-Directed
    Chemical Substances Glutamic Acid (3KX376GY7L) ; Coloring Agents ; Peroxidases (EC 1.11.1.-)
    Language English
    Publishing date 2024-04-08
    Publishing country Germany
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2020469-3
    ISSN 1439-7633 ; 1439-4227
    ISSN (online) 1439-7633
    ISSN 1439-4227
    DOI 10.1002/cbic.202300872
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag.

    Abd Elhameed, Heba A H / Hajdu, Bálint / Balogh, Ria K / Hermann, Enikő / Hunyadi-Gulyás, Éva / Gyurcsik, Béla

    Protein expression and purification

    2019  Volume 159, Page(s) 53–59

    Abstract: The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the ... ...

    Abstract The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3'-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.
    MeSH term(s) Amino Acid Sequence ; Bacterial Proteins/chemistry ; Chromatography, Affinity/methods ; Cloning, Molecular ; Colicins/chemistry ; Colicins/genetics ; Escherichia coli/metabolism ; Histidine/chemistry ; Oligopeptides/chemistry ; Peptide Hydrolases/chemistry ; Peptide Hydrolases/genetics ; Protein Processing, Post-Translational ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics
    Chemical Substances Bacterial Proteins ; ColE7 protein, E coli ; Colicins ; His-His-His-His-His-His ; Oligopeptides ; Recombinant Proteins ; Histidine (4QD397987E) ; Peptide Hydrolases (EC 3.4.-)
    Language English
    Publishing date 2019-03-22
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1055455-5
    ISSN 1096-0279 ; 1046-5928
    ISSN (online) 1096-0279
    ISSN 1046-5928
    DOI 10.1016/j.pep.2019.03.009
    Database MEDical Literature Analysis and Retrieval System OnLINE

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