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Resistance to macrolides, lincosamides and streptogramins in coagulase-negative staphylococci in ČR
Novotná, Gabriela ; Janata, Jiří (advisor) ; Petráš, Petr (referee) ; Sigler, Karel (referee)
VÝSLEDKY Analýza okolí genu msr(A) 45 součástí kompletního transpozonu Tn552 a s rekombinačním místem resbinR je identická až k místu rekombinace (resbinR site I). Druhá, v pozici JCSC1435 261884-2618311, je součástí sekvence transpozonu Tn5404. Kompletní transpozon Tn5404 je na vnějších okrajích ohraničen 7 bp dlouhou přímou repeticí (AGTAACT), jež vznikla zdvojením místa inzerce transpozonu (Obr. 5.3.), stejné inzerční místo pro Tn552 bylo pozorováno v chromozómu Entereococcus faecalis CH116 (146, 155). Shrnutí výsledků: Gen msr(A) byl u 41 izolátů lokalizován na chromozómu ve společném místě, pouze u dvou izolátů byl gen lokalizován na plazmidu. Oblast chromozomálně kódovaného genu msr(A) je u všech patnácti podrobněji studovaných izolátů (Tab. 5.1.) vysoce homologní s plazmidem πSh1, integrovaným v genomu S. haemolyticus JCSC1435 izolovaného v Japonsku (179). Uspořádání genů na tomto plazmidu je konzervovaným souborem jednotlivých genových motivů, jehož menší fragmenty byly odděleně popsány v několika dřívějších studiích. Rozdíly v msr(A) hybridizačních profilech jsou dány výhradně variabilitou v úseku kódujícím resolvasu Bin a rekombinasu Sin. Nalezli jsme pět různých typů uspořádání msr(A) oblastí, které se liší od JCSC1435 tímto: i) vložením části transpozonu Tn552 (typ KM50; 7,2kb msr(A)...
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Dimerization of Streptococcus pneumoniae eukaryotic-type SerThr protein kinase and characterization of its substrate, phosphoglucosamine mutase GlmM
Pallová, Petra ; Branny, Pavel (advisor) ; Janata, Jiří (referee) ; Španová, Alena (referee)
104 7 Závěr 7.1 Proteinkinasa StkP Připravili jsme kmen S. pneumoniae KDTM-his, který ve svém genomu kóduje epitopem značenou kinázovou doménu proteinkinasy StkP ukotvenou do membrány pomocí transmembránové domény. Imunologickou detekcí s monoklonální protilátkou proti histidinové kotvě jsme potvrdili lokalizaci proteinu v membránové frakci S. pneumoniae. V in vitro kinázových reakcích jsme prokázali, že se jedná o plně funkční protein s autofosforylační aktivitou. Pomocí in vivo reportérového systému jsme zjistili, že transmembránová doména a extracelulární doména proteinkinasy StkP tvoří stabilní dimery. Dimerizace proteinkinasy StkP byla následně potvrzena pomocí nativní elektroforézy. Je tedy velmi pravděpodobné, že proteinkinasa StkP se in vivo vyskytuje ve formě dimeru a dimerizace je nutným předpokladem její autofosforylační aktivity. Na základě těchto výsledků jsme vyslovili hypotézu, že protein kódující kinázovou doménu postrádající transmembránovou doménu není funkční, neboť není schopen dimerizace a podléhá degradaci. 7.2 Fosfoglukosaminmutasa GlmM V in vitro kinázové reakci na bezbuněčných lyzátech S. pneumoniae Cp1015, ∆stkP a ∆phpP-stkP v přítomnosti rekombinantní proteinkinasy StkP jsme prokázali fosforylaci proteinu o molekulové hmotnosti odpovídající fosfoglukosaminmutase GlmM....
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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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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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