Struktura a funkce flavinreduktas

Bc. Dominik Banáš

Bachelor's thesis

Structure and function of flavinreductases

Abstract:

Flavinreductases, either alone or in co-operation with monooxygenases, create a very important part of the enzymatic machinery of redox metabolism in any cell. Understanding structures and functions of these enzymes deepens biochemical knowledge of cellular metabolism and finds practical application in biotechnology. In this bachelor thesis, we have focused on the protein product of the gene Pden_5119 from the bacterium Paracoccus denitrificans in terms of its protein-protein interactions. Protein C-5119 was immobilized on a Ni-IDA resin and incubated with cytosol of the strain Paracoccus denitrificans 1222, grown in the presence or absence of methylviologen, a redox stressor. Both incubations were realized in the presence of FMN. Eluates were analyzed by SDS-PAGE, unfortunately mass spectrometry can’t be realized because of technical reasons. Fluorescence anisotropy measurements were made to find out if monooxygenases C-5013 and N-5007 interact with flavinreductase C-5119. The monooxygenases were also immobilized on Ni-IDA resin in order to detect potential proteinprotein interactions by a pull-down approach similarly as in the case of the protein C-5119.

Abstract:

Flavínreduktázy, či už samy alebo v kooperácii s monooxygenázami, tvoria veľmi dôležitú súčasť enzymatickej mašinérie redoxného metabolizmu ktorejkoľvek bunky. Porozumenie štruktúrnych a funkčných aspektov týchto enzýmov prehlbuje biochemické znalosti o bunečnom metabolizme a nachádza svoje praktické uplatnenie v biotechnológiach. V tejto bakalárskej práci sme sa zamerali na produkt génu Pden_5119 z baktérie Paracoccus denitrificans z hľadiska jeho interakcií s inými proteínmi. Proteín C-5119 bol imobilizovaný na Ni-IDA nosič a inkubovaný s cytosolom baktérii Paracoccus denitrificans kmeň 1222, ktoré rástli v prítomnosti alebo absencii metylviologénu, ktorý pôsobí ako redoxný stresor. Obe kultivácie prebiehali v prítomnosti FMN. Eluáty boli analyzované pomocou SDS-PAGE, hmotnostnú spektrometriu nebolo možné z technických príčin zrealizovať. Bolo prevedené meranie fluorescenčnej anizotropie, aby sa zistilo či monooxygenázy C-5013 a N-5007 interagujú s flavínreduktázou C-5119. Monooxygenázy boli taktiež imobilizované na Ni-IDA nosič, aby sa detekovali prípadné proteínové interakcie metódou pull-downu podobne ako v prípade proteínu C-5119.

Keywords

flavínreduktázy, monooxygenázy, proteín-proteínové interakcie, flavoenzymológia, Pden_5119, Pden_5013, Pden_5007, flavinreductases, monooxygenases, protein-protein interactions, flavoenzymology

Language used: Slovak

Date on which the thesis was submitted / produced: 10. 5. 2017

Thesis defence

• Date of defence: 13. 6. 2017
• Supervisor: prof. RNDr. Igor Kučera, DrSc.

Masaryk University

Faculty of Science

Bachelor programme / field:
Biochemistry / Biochemistry