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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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Molecular dynamics of proteins interacting with substrate or ligand: mitochondrial processing peptidase and FixL oxygen sensor
Dvořáková Holá, Klára ; Janata, Jiří (advisor) ; Váchová, Libuše (referee) ; Branny, Pavel (referee)
I I Ovenvrew I I I II I I I I I I I I I The presenteddoctoralthesis includesfive publishedscientificarticlesand one manuscriptpreparedfor submission.All describestudieson threeproteinmodels.The four papersnumbered(1)- (4)inthelistofpublications,sharea commonobjectivee.i.observingand describingfunctionalproteindynamicsandconformationchangeinducedby ligandor substrate binding,andrepresentthemainresultofmyPhDwork. Thepapers(1)and(4)offerresultsof a projectfrommyhomelaboratoryattheInstitute ofMicrobiologyAS CR, LaboratoryforBiologyofSecondaryMetabolism,underthesupervisionof JiřÍ Janata, PhD. The projectis focusedon the protein-proteindynamicsinteractionof mitochondrialprocessingpeptidase(MPP) fromSaccharomycescerevisiaewithits preproteins substrates. The papers(2)and(3)describeresultsof a project,inwhichI haveparticipatedduring myMarieCuriefellowshipattheEcolePolytechnique(PalaiseauCedex,France),Laboratoryfor OpticsandBiosciences,in2004.Theprojectconcernsresearchon proteinstructuraldynamicsof theheme-basedoxygensensorFixLfromBradyrhizobiumjaponicum,inwhichoxygenbindingto the heme sensor domain inducesconformationchange,which regulatesthe activityof neighboringkinasedomain. ln bothprojects,analogyin methodicalapproach,i.e.seriesof molecularbiologyand...
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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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Analysis of lincomycin biosynthetic genes usable for the preparation of hybrid compounds
Ulanova, Dana ; Janata, Jiří (advisor) ; Varečka, Ľudovít (referee) ; Svobodová, Jaroslava (referee)
Analysis of lincomycin biosynthetic genes usable for the preparation of hybrid compounds Lincosamide antibiotics have relatively simple molecular structure compared with other types of antibiotics. However, these molecules contain so called "hot spots" whose modification leads to the development of more effective drugs. Combinatorial biosynthesis appears to be a promising way to design and preparation of lincosamides' derivatives. For this purpose a perfect knowledge of the natural biosynthetic pathway is needed. The submitted doctoral thesis analyzed genes lmbIH, K, N, Q, T, U and V with unknown function from the lincomycin biosynthetic gene cluster. Mutation analysis of lmbN has proven its bifunctional character, 5 'end of the gene is coding for a potential N-demethyllincomycin synthetase subunit, while the rest of the gene encodes an enzyme for synthesis of sugar precursor methylthiolincosamide (MTL). Gene lmbT was also assigned to the biosynthesis of the sugar precursor. In the case of other genes the determination of function was not so clear. It is assumed that their roles in the biosynthetic pathway are more regulatory or supporting. The mutasynthetic approach was used for the preparation of lincomycin derivatives with longer alkyl residues. Activity of obtained lincomycin derivatives was tested on a...
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Study of the key points of lincomycin and celesticetin biosynthesis
Vobruba, Šimon ; Janata, Jiří (advisor) ; Bobek, Jan (referee) ; Kutejová, Eva (referee)
Lincosamides form a small but important group of specialized microbial metabolites with antibiotic activity. The most important members of this group are celesticetin and clinically used lincomycin. Structurally, lincosamides are composed of an amino sugar and an amino acid connected by an amide bond. The amino acid precursors of both lincosamides remarkably differ. Proteinogenic L-proline is the precursor of celesticetin, while an unusual amino acid (2S,4R)-4-propyl- L-proline (PPL) is incorporated in the more efficient compound lincomycin. Surprisingly, both these precursors are recognized and activated for further biosynthetic steps by homologous adenylation domains CcbC and LmbC, respectively. The detailed description of this amino acid recognition and activation step, which is critical for the biological activity of the resulting compound, was the aim of the first part of this thesis. The site-directed mutagenesis of the LmbC substrate binding pocket and biochemical characterization of resulting mutants were employed to identify the residues crucial for the activation of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket (like in CcbC) to the PPL-specific enzyme (LmbC). The substitution of only three amino acid...
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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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