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Lessons from nature - preparation of hybrid bioactive compounds
Vobruba, Šimon ; Janata, Jiří (advisor) ; Zikánová, Blanka (referee)
Secondary metabolites are biologically active compounds produced mainly by microorganisms. They are not essential for survival of producing strains, however, they significantly affect their physiology and ecology. They are frequently used in pharmacology, biology and chemistry. The present work describes the current state of knowledge concerning origin and evolution of secondary metabolites. The secondary metabolites biosynthetic genes are usually organised in clusters. The basic mechanisms of secondary metabolite gene clusters modification are gene mutations or intragenic rearrangements. These mechanisms are typically involved in natural evolution of gene clusters coding for secondary metabolites with modular type of biosynthesis. The subclusters of different origin can also fuse to form a new hybrid compound biosynthetic gene cluster. Similar evolutionary event probably occurred also in case of biosynthesis of two model groups of natural compounds - lincosamides and pyrrolobenzodiazepines. Analogous approaches are used in genetic engineering to construct producers of new more efficient bioactive compounds. Examples of such genetic modifications of gene clusters involved in the biosynthesis of compounds from nonribosomal peptides, polyketides and lincosamides groups are described. Possible future...
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The substrate specificity of adenylation domains of synthetases in secondary methabolism.
Vobruba, Šimon ; Janata, Jiří (advisor) ; Fišer, Radovan (referee)
The crucial part of the biosynthesis of lincosamide antibiotics lincomycin and celesticetin is the condensation of amino sugar and amino acid moieties. This reaction is catalysed by the oligomeric enzyme lincosamide synthetase (LS). One of the most important components of LS is adenylation domain recognizing and activating amino acid precursor. The substrate specificity of adenylation domain is determined by "nonribosomal code", 10 amino acids residues which side chains are in close contact with the activated substrate. The homologous adenylation domains LmbC from biosynthesis of lincomycin and CcbC from biosynthesis of celesticetin exhibit strong substrate specificity for their natural substrates (2S,4R)-4-propyl-L-proline (PPL) and L-proline, respectively. At first the effect of selected amino acid residues of LmbC nonribosomal code on the substrate specificity of the whole domain was tested. The amino acids residues, most important for preference of PPL substrate over L proline, were determined: G308, A207 and L246. Then the effect of double mutations in nonribosomal codes of both LmbC and CcbC on their substrate specificity was evaluated. The double mutants LmbC G308V + A207F and CcbC V306G + F205A were prepared and tested biochemically. The results brought new evidence of validity of homologous models...
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Lessons from nature - preparation of hybrid bioactive compounds
Vobruba, Šimon ; Janata, Jiří (advisor) ; Zikánová, Blanka (referee)
Secondary metabolites are biologically active compounds produced mainly by microorganisms. They are not essential for survival of producing strains, however, they significantly affect their physiology and ecology. They are frequently used in pharmacology, biology and chemistry. The present work describes the current state of knowledge concerning origin and evolution of secondary metabolites. The secondary metabolites biosynthetic genes are usually organised in clusters. The basic mechanisms of secondary metabolite gene clusters modification are gene mutations or intragenic rearrangements. These mechanisms are typically involved in natural evolution of gene clusters coding for secondary metabolites with modular type of biosynthesis. The subclusters of different origin can also fuse to form a new hybrid compound biosynthetic gene cluster. Similar evolutionary event probably occurred also in case of biosynthesis of two model groups of natural compounds - lincosamides and pyrrolobenzodiazepines. Analogous approaches are used in genetic engineering to construct producers of new more efficient bioactive compounds. Examples of such genetic modifications of gene clusters involved in the biosynthesis of compounds from nonribosomal peptides, polyketides and lincosamides groups are described. Possible future...
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Evolution and expression of the Odorant Binding Proteins in selected species of mice
Vinkler, David ; Stopka, Pavel (advisor) ; Trachtulec, Zdeněk (referee)
Odorant-binding proteins (OBPs) are small soluble proteins expressed at high levels in the proximity of olfactory receptors. OBPs act as solubilizers and carriers of the lipophilic odorants in the aqueous mucus of mammals and other vertebrates. OBPs have now been studied nearly thirty years, but in comparison to the wealth of data available on their structural chemistry and molecular biology, our knowledge about gene expression and function of these proteins is still insufficient. This work provides new insights into the tissue specificity of OBP and presents several new sequences of genes governing these proteins in selected species of mice.
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