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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We investigated the role of epiQ in the biosynthesis of the lantibiotic epidermin. epiQ was essential for epidermin production. It was shown that EpiQ controls epidermin production by transcriptionally activating the epiA promoter, used for transcription of most of the epidermin biosynthetic genes. Additional copies of epiQ increased epidermin production in the epidermin-producing wild-type strain Staphylococcus epidermidis Tü3298. The epiA promoter region was characterized by primer extension analysis. Two inverted repeats, putative operator sites for EpiQ binding, are located upstream of the −35 region and one is localized downstream of the-10 region. Crude protein extracts from S. epidermidis Tü3298 and epiQ expressing Escherichia coli cells led to gel mobility shifts of a DNA fragment bearing the inverted repeat which is located immediately upstream of the -35 region. DNA fragments bearing the other two inverted repeats were not shifted. The epiQ gene product could be detected by overexpression in the E. coli T7 system using antiserum raised against synthetic pep-tides of EpiQ. Furthermore, EpiQ, like other DNA-binding proteins, was shown to bind strongly to heparin sepharose.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 153 (1997), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The Staphylococcus epidermidis genes encoding the His-tag-labelled epidermin precursor peptide EpiA and the flavoenzyme EpiD or the mutant protein EpiD-G93D, which lacks the coenzyme, were co-expressed and the proteins were synthesized in vivo in Escherichia coli. Only in the presence of EpiD was the precursor peptide converted to a reaction product with a decrease in mass of 44–46 Da. This result confirms the in vitro experiments carried out with purified EpiA and purified EpiD from Staphylococcus epidermidis [Kupke et al. (1994) J. Biol. Chem. 269, 5653–5659]. EpiD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue of EpiA to a [Z]-enethiol structure. In the presence of EpiD, the amount of purified (modified) peptide EpiA was several-fold higher than in the presence of EpiD-G93D, indicating that the stabilization of EpiA against proteolysis is due to an interaction with EpiD or to the presence of the C-terminal modification. The presented experimental approach will be valuable for the analysis of enzymes that catalyze posttranslational modification reactions of peptides and proteins.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R−1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A−4-E-P-R−1- to -A−4-E-P-Q−1- by site-directed mutagenesis.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1572-9699
    Keywords: Biosynthesis of lantibiotics ; epidermin ; epilancin-K7 ; gallidermin ; pep5
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Antonie van Leeuwenhoek 69 (1996), S. 139-150 
    ISSN: 1572-9699
    Keywords: α, β-dehydroamino acids ; D-amino acids ; flavoprotein ; oxidative decarboxylation ; post-translational modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Several newly reported post-translational modification reactions are involved in lantibiotic biosynthesis. A short overview of the present knowledge on the post-translational modifications and on the enzymes involved in lantibiotic biosynthesis is given. The oxidative decarboxylation of the epidermin precursor peptide EpiA is described in detail. The FMN-containing oxidoreductase EpiD is involved in the formation of the C-terminal S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin: under reducing conditions the side chain of the C-terminal cysteine residue of EpiA is converted to an enethiol. EpiD has no absolute substrate specificity and can be used for modification of peptides having the C-terminal consensus motif [V/I/L/(M)/F/Y/W]-[A/S/V/T/C/(I/L)]-C.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1572-9699
    Keywords: protein engineering ; lantibiotics ; expression systems ; nisin ; epidermin ; Pep5
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 0044-8249
    Keywords: Chemistry ; General Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 0570-0833
    Keywords: biosyntheses ; decarboxylations ; epidermin ; peptides ; sulfur compounds ; Chemistry ; General Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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