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Impact of the glycine-rich loop on the function of processing peptidases of the mitochondrial type
Kučera, Tomáš ; Janata, Jiří (advisor) ; Bařinka, Cyril (referee) ; Ettrich, Rüdiger (referee)
The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...
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Function of LmbW protein in biosynthesis of antibiotic lincomycin
Steiningerová, Lucie ; Janata, Jiří (advisor) ; Seydlová, Gabriela (referee)
4-Alkyl-L-proline derivatives (APD) are specialized precursors involved in the biosynthesis of at least three groups of different natural compounds: some pyrrolo-1,4-benzodiazepines with antitumor activity, bacterial hormone hormaomycin and clinically used lincosamide antibiotic lincomycin. These compounds share a biosynthetic pathway encoded by 5 or 6 homologous genes present in the biosynthetic gene clusters of the producing organisms. Similarities in biosynthesis and differences between APD structures of these compounds could be used to prepare a hybrid producing strain of biologically more effective lincomycin derivative. Unusual amino acid 4-propyl-L-proline (PPL) is the APD precursor of lincomycin. The originally proposed scheme of the PPL pathway does not comply with our current knowledge. Therefore, it was necessary to revise this scheme according to new results. The first two steps of the PPL pathway are functionally proved. Probing the next step was the main aim of this work. The protein LmbW was overproduced and its methyltransferase activity was confirmed in vitro. LmbW is able to directly methylate intermediate of second step of the pathway while the originally scheme proposed methylation at a later stage of biosynthesis. LmbW is also able to attach a longer alkyl chain to its substrate. This...
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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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Common features of the biosynthetic pathways of lincomycin,some pyrrolo-1,4-benzodiazepines and hormaomycin
Steiningerová, Lucie ; Janata, Jiří (advisor) ; Mašek, Tomáš (referee)
Lincosamides, pyrrolo-1,4-benzodiazepines (PBD) and hormaomycin are biologically active substances differing in their structure and mode of action. Lincosamides lincomycin and clindamycin are clinically used antibiotics, PBD exhibit antitumor activity and hormaomycin is a bacterial hormone. Unusual precursor, 4-propyl-L-proline (PPL) derived from L-tyrosine is incorporated in the lincomycin structure. Surprisingly, structurally and functionally dissimilar hormaomycin and some PBD (tomaymycin, anthramycin, sibiromycin) incorporate L-proline derivative with two- or three- carbon side chain of the same biosynthetic origin. Accordingly, a set of orthologous genes coding for biosynthesis of these structurally similar precursors has been found in appropriate biosynthetic gene clusters. There is a clear evolutionary relationship among the biosynthesis of lincomycin, hormaomycin and some PBD. However, the evolutionary origin of PPL pathway is not known as well as its further development. Only a limited number of naturally occured modifications of the pathway has been described and the history of supposed horizontal gene transfers of biosynthetic genes remains unknown. Answers to these questions are particularly important due to the preparation of new hybrid substances with improved bioactive effects using methods...
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Preparation and characterization of LmbX protein involved in lincomycin biosynthesis
Jiráčková, Petra ; Janata, Jiří (advisor) ; Janderová, Blanka (referee)
Lincomycin is an antibiotic used in clinical praxis. It is produced by Streptomyces lincolnensis. Lincomycin is composed of an amino-sugar and an amino-acid moiety linked by an amide bond. The amino-acid precursor is propylproline (PPL), whose biosynthesis undergoes the pathway derived from tyrosine. The modified PPL biosynthesis pathway was also discovered in pyrrolobenzodiazepines (PBD) and hormaomycin. In the biosynthesis of PBD the PPL precursor is further modified by reactions catalysed by specific enzymes missing in the biosynthesis of lincomycin. The genes encoding these enzymes could be transferred to the lincomycin biosynthetic gene cluster. In this way we could get producers of hybrid antibiotics with better properties and even antimalaric effects. Six enzymes participate in PPL biosynthesis, which are encoded in the lincomycin biosynthetic gene cluster. The first two reactions of PPL biosynthesis pathway are proven, therefore, this work focuses on the third reaction that is supposed to be catalysed by protein LmbX according to literature. The proposed function of LmbX is a hydrolysis of C-C bond. However, LmbX belongs to the protein family of isomerases by sequence homology. The protein LmbX was overproduced in this work and its activity was tested in the presence of the expected...
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Characterization of gycin-rich loop of mitochondrial processing peptidase alpha subunit from Saccharomyces cerevisiae
Chytrá, Dana ; Janata, Jiří (advisor) ; Zikánová, Blanka (referee)
Mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which belongs to M16B subfamily of metaloendopeptidases. A universal function of this enzyme is in recognition and cleavage of great number of mitochondrial preprotein presequences, which differ in length and amino acid sequence. MPP consists of catalytical β-MPP and probably recognizing α-MPP. The most conservative region in α-MPP is GRL - glycine-rich loop. Its function is supposed in primary interaction with preprotein presequence. It is possible to study conformational change of GRL after binding the substrate by fluorescence experiments. In this diploma thesis the constructs coding the α-MPP with the single reporter tryptophan residue in the position 289 or 299 were prepared using site-directed mutagenesis. These forms of α-MPP were produced in E. coli BL21(DE3)+pGroESL. Activities of MPP dimer containing α-MPP with the single tryptophan residue in the reporter position were compared with MPP from wild type of S. cerevisiae. Used substrate was yeast malate dehydrogenase precursor with fused presequence (pMDH) from three organisms (yeast, mouse and melon). These presequences differ in their length. Activities of MPP dimer containing α-MPP with the single reporter tryptophan residue in the position 289 were about 70 % while...
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Comparative analysis of celesticetin and lincomycin biosynthetic gene clusters
Koběrská, Markéta ; Janata, Jiří (advisor) ; Lichá, Irena (referee) ; Kormanec, Ján (referee)
Comparative analysis of celesticetin and lincomycin biosynthetic gene clusters Markéta Koběrská PhD thesis 2010 The introductory part of the thesis presents isolation and sequencing of lincomycin gene cluster from type strain Streptomyces lincolnensis ATCC 25466. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin industrial strain Streptomyces lincolnensis 78-11. Analysis of the cluster flanking regions revealed its localization within the genome of S. lincolnensis ATCC 25466. The cluster-bearing cosmid was integrated into the chromosome of lincomycin non-producing strains Streptomyces coelicolor CH 999 and Streptomyces coelicolor M 145. The modified strains heterologously produced lincomycin, but the level dropped to approximately 1-3% of the production in S. lincolnensis ATCC 25466. The exact sequence of lincomycin gene cluster from the type strain allowed isolation and sequence analysis of the gene cluster of structurally related celesticetin. The analysis revealed 24 putative genes, 18 of them homologous with the genes participating in lincomycin biosynthesis. Four celesticetin specific genes are encoding enzymes involved in the salicylate biosynthesis and attachment, one is coding for...
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Genetic characterization of tetracycline resistance in selected soil isolates of bacteria.
Hucková, Tereza ; Lichá, Irena (advisor) ; Janata, Jiří (referee)
GENETIC CHARACTERIZATION OF TETRACYCLINE RESISTANCE IN SELECTED SOIL ISOLATES OF BACTERIA Abstract Antibiotic resistance is becoming increasingly widespread among bacterial organisms. To respond to the situation it is necessary to understand in detail all the mechanisms of resistance as well as expansion possibilities of resistance genes in the environment. In this study we attempted to identify tetracycline resistance determinants in selected soil gram-positive and gram-negative isolates. The isolates originate from unfertilized soil and from soil fertilized with tetracycline-contaminated manure. We tested the soil isolates for the presence of twenty three selected tetracycline resistance determinants and presence of tetracycline resistance determinants in DNA libraries. We identified one of the tetracycline resistance determinants tet(K) in Staphylococcus sp. A DNA fragment was amplified with primers for tet(M) determinant in Arthrobacter sp., but its presence was not confirmed by the sequence analysis. None of the tested tetracycline resistance determinants were present in the genera Chryseobacterium and Stenotrophomonas. However, we managed to prove the ongoing horizontal gene transfer between the genus Stenotrophomonas and the genus Chryseobacterium. The transferred sequences encoded hybrid protein and...
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