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The basis of the structural-functional study of selected enzymes from the 4-alkyl- L-proline pathway
Zachovalová, Veronika ; Zdvořáková, Lucie (advisor) ; Košek, Dalibor (referee)
4-alkyl-L-proline derivate (APD) is building block of some important, bioactive specialized metabolites produced by Actinobacteria. It is found, for example, in the structure of lincosamide antibiotic lincomycin, bacterial hormone hormaomycin having antimicrobial activity, the antituberculosis compound griselimycin and pyrrolo-1,4-benzodiazepins (PBD) which have anticancer effects. APDs, or their precursors, are formed from L-leucine or L-tyrosin in a unique biosynthetic pathway consisting of up to six homologous proteins named Apd1 - Apd6. The first part of this thesis lays the foundation for a crystallization study of SAM-dependent methyltransferase Apd3 from the biosynthesis of lincomycin - the protein LmbW, in order to elucidate the identity of its natural substrate, which remains up to date a subject of scientific discussions. A purification method providing LmbW protein of sufficient quantity, quality and purity for crystallization was optimized. Subsequently the crystallization conditions allowing crystal growth were defined. The second part of the thesis deals with the elucidation of molecular basis of different reaction specificity of homologous F420H2-dependent oxidoreductases, Apd6. They catalyse reduction of identical substrate with two conjugated double bonds, however, some of them...
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Analysis and isolation of intermediates involved in the biosynthetic pathway of alkylproline derivatives
Cudlman, Lukáš ; Bosáková, Zuzana (advisor) ; Michalíková, Klára (referee)
(EN) This work aims at preparation, analysis and isolation of intermediates of biosynthetic pathways of 4-alkyl-L-proline derivatives for their structural elucidation. Compounds with incorporated 4-alkyl-L-proline derivatives include clinically used lincosamide antibiotic, lincomycin A, antitumor pyrrolobenzodiazepines and bacterial hormone hormaomycin. Detailed knowledge of biosynthetic pathways of these biological active substances can be used to prepare new, more efficient derivatives. The first part of this work focuses on yellow-coloured dicarboxylic intermediates 1 and 2 of the biosynthetic pathway of 4-propyl-L-proline - the precursor of lincomycin A. In the presence of the methylation agent, S-adenosyl-L-methionine, and LmbW C- -methyltransferase, 1 was partially converted into intermediate 2. Using ultra-high performance liquid chromatography, both intermediates were identified from absorption and mass spectrometry spectra. A semi-preparative chromatographic method for isolation of both intermediates was developed. Surprisingly, a significantly lower stability of 2 compared to intermediate 1 was observed in an in vitro enzymatic reaction mixture. The second part of the work focuses on 4-ethylidene-L-proline - the precursor of tomaymycin belonging to pyrrolobenzodiazepines. After...
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The Shared Steps of Lincomycine and Pyrrolo-benzodiazepine Biosynthetic Pathways.
Jiráčková, Petra ; Janata, Jiří (advisor) ; Fišer, Radovan (referee)
Lincomycin and its semi-synthetic derivate clindamycin are therapeutically used antibiotics inhibiting protein synthesis in sensitive bacteria. Their structure is composed of an amino-sugar and an amino-acid moiety, which are linked with an amide bond. Some time ago the sequence of lincomycin-production gene cluster was established and functions of the most proteins coded from gene cluster were proposed on the basis of a comparison with known proteins and gene-inactivations. For a long time it was clear that the amino-acid moiety of linkomycin (propylproline) is related to a dihydropyrrol moiety of pyrrolo[1,4]benzodiazepine (PBD). Both of these moieties are derived from L-tyrosine and they undergo the same biosynthetic pathway at first. But other biosynthetic steps divide. It is believed that a transfer of genes from the PBD-production gene cluster to the lincomycin-production gene cluster could modify the structure of propylproline and improve traits of lincomycin e.g. increase antimalaric effects. The most effective antibiotics are alkyl derivatives, their chemical synthesis is very complicated, but they could be prepared via genetic engineering as so-called hybrid antibiotics.
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The role of F₄₂₀H₂-dependent reductases in the biosynthesis of microbial bioactive metabolites incorporating a 4-alkyl-˪-proline derivate
Steiningerová, Lucie
Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...
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Biosynthesis of propylproline building unit of lincomycin
Jirásková, Petra ; Janata, Jiří (advisor) ; Čejková, Alena (referee) ; Fišer, Radovan (referee)
The clinically used antibiotic lincomycin consists of an amino-sugar and an amino-acid moiety. The incorporated amino-acid 4-propyl-L-prolin (PPL) is very important for the linomycin bioactivity, as evidenced by the lower activity of the related antibiotic celesticetin, which incorporates proteinogenic L-prolin instead. Gene clusters for the biosynthesis of both lincosamides are published and reflect a common basis - biosynthesis of amino-sugar precursor and condensation reactions. Additionally, in the biosynthetic gene cluster for lincomycin there is a sub-cluster of genes encoding the biosynthesis of PPL, the alkylated proline derivative (APD). PPL has a common biosynthetic origin with other APDs that are part of the structures of antitumor pyrrolobenzodiazepines and the signal molecule hormaomycin, which is also reflected in the presence of homologous genes in their gene clusters. The acquired knowledge on PPL biosynthesis thus can be applied to a larger group of natural products. The first overall concept of APD biosynthesis was published forty years ago. The milestone was the year 1995 when the gene cluster for lincomycin biosynthesis was published and specific gene products have been proposed for individual biosynthetic steps. The functional proof of proteins has been performed so far just...
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The role of F₄₂₀H₂-dependent reductases in the biosynthesis of microbial bioactive metabolites incorporating a 4-alkyl-˪-proline derivate
Steiningerová, Lucie ; Janata, Jiří (advisor) ; Masák, Jan (referee) ; Obšilová, Veronika (referee)
Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...
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Analysis and isolation of intermediates involved in the biosynthetic pathway of alkylproline derivatives
Cudlman, Lukáš ; Bosáková, Zuzana (advisor) ; Michalíková, Klára (referee)
(EN) This work aims at preparation, analysis and isolation of intermediates of biosynthetic pathways of 4-alkyl-L-proline derivatives for their structural elucidation. Compounds with incorporated 4-alkyl-L-proline derivatives include clinically used lincosamide antibiotic, lincomycin A, antitumor pyrrolobenzodiazepines and bacterial hormone hormaomycin. Detailed knowledge of biosynthetic pathways of these biological active substances can be used to prepare new, more efficient derivatives. The first part of this work focuses on yellow-coloured dicarboxylic intermediates 1 and 2 of the biosynthetic pathway of 4-propyl-L-proline - the precursor of lincomycin A. In the presence of the methylation agent, S-adenosyl-L-methionine, and LmbW C- -methyltransferase, 1 was partially converted into intermediate 2. Using ultra-high performance liquid chromatography, both intermediates were identified from absorption and mass spectrometry spectra. A semi-preparative chromatographic method for isolation of both intermediates was developed. Surprisingly, a significantly lower stability of 2 compared to intermediate 1 was observed in an in vitro enzymatic reaction mixture. The second part of the work focuses on 4-ethylidene-L-proline - the precursor of tomaymycin belonging to pyrrolobenzodiazepines. After...
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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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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 N-demethyllincomycin-methyltransferase.
Poľan, Marek ; Najmanová, Lucie (advisor) ; Petříčková, Kateřina (referee)
Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...
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