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Charakterizácia N-demetyllinkomycin-metyltransferázy.

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Title:
Charakterizácia N-demetyllinkomycin-metyltransferázy.
Title (in czech):
Charakterizace N-demetyllinkomycin-metyltransferázy.
Titile (in english):
Characterization of N-demethyllincomycin-methyltransferase.
Type:
Diploma thesis
Author:
Mgr. Marek Poľan
Supervisor:
Mgr. Lucie Najmanová, Ph.D.
Opponent:
Mgr. Kateřina Petříčková, Ph.D.
Thesis Id:
60806
Faculty:
Faculty of Science (PřF)
Department:
Department of Genetics and Microbiology (31-140)
Study programm:
Biology (N1501)
Study branch:
Microbiology (NMIKRO)
Degree granted:
Mgr.
Defence date:
20/09/2010
Defence result:
Very good
Language:
Slovak
Keywords (in czech):
linkomycín, metyltransferáza, LmbJ, antibiotiká, biosyntetický zhluk, enzýmová aktivita
Keywords:
lincomycin, methyltransferase, LmbJ, antibiotics, biosynthetic cluster, enzyme kinetics
Abstract (in czech):
Linkomycín je prírodne antibiotikum zaradené do skupiny linkozamidových antibiotík, ktorého zhluk biosyntetických génov je známy a funkcia mnohých génov z tohto zhluku už bola popísaná. Táto práca je zameraná na štúdium záverečného kroku bisyntetickej dráhy linkomycínu, metyláciu na dusíku pyrolového reťazca propylprolínovej podjednotky N-demetyllinkomycínu (NDL). Cieľom práce bolo charakterizovať enzým LmbJ, katalyzujúci tento biosyntetický krok. Všetky stanovenia boli uskutočnené pre enzým LmbJ s histidínovou kotvou, ktorý bol pripravený heterológnou expresiou v bunkách Escherichia coli. Bolo stanovené teplotné a pH optimum metylačnej reakcie, rovnako ako Michelisove konštanty pre oba substráty reakcie: N-demetyllinkomycín a S-adenozylmetionín (SAM - donor metylovej skupiny). S výnimkou pH optima se všetky stanovené parametre výrazne odlišovali od publikovaných údajov pre enzým izolovaný z prirodzeného zdroja. Na základe porovnania výsledkov elektrónovej mikroskopie, natívnej elektroforézy a gélovej filtrácie bol vytvorený predpokladaný model kvartérnej štruktúry enzýmu. Na rozdiel od väčšiny doteraz popísaných metyltransferáz, ktoré sa vyskytujú v monomérnej, dimérnej alebo tetramérnej forme, sa v našom prípade jedná s najväčšou pravdepodobnosťou o hexamér.
Abstract:
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 monomeric, dimeric or tetrameric form. By contrast in our case the most probable model of quarternary structure is a hexamer.
Documents
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Download Text of the thesis Mgr. Marek Poľan 2.47 MB
Download Abstract in czech Mgr. Marek Poľan 42 kB
Download Abstract in english Mgr. Marek Poľan 42 kB
Download Supervisor's review Mgr. Lucie Najmanová, Ph.D. 1.05 MB
Download Opponent's review Mgr. Kateřina Petříčková, Ph.D. 1.28 MB
Download Defence's report 228 kB