Istraživanje supstratne specifičnosti acil-CoA tioesteraze I iz bakterije Pseudomonas aeruginosa korištenjem simulacije molekularne dinamike

master's thesis

• Title (english): Investigation of substrate specificity of acyl-CoA thioesterase I from the bacteria Pseudomonas aeruginosa using molecular dynamics
• Author: Saša Petrović
• Mentor: Branimir Bertoša (mentor)
• Committee member: Branimir Bertoša (predsjednik povjerenstva)
• Committee member: Ita Gruić Sovulj (član povjerenstva)
• Committee member: Kristian Vlahoviček (član povjerenstva)
• Committee member: Dijana Škorić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2013-09-19, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Acil-­CoA-­tioesteraza I (TesA) iz bakterije Pseudomonas aeruginosa lipolitički je enzim koji pripada obitelji GDSL hidrolaza. Obitelj GDSL hidrolaza relativno je nova i neistražena skupina enzima s velikim potencijalom za primjenu u farmaceutskoj i prehrambenoj industriji, kao i za daljnja biokemijska istraživanja. Posebnost ove enzimske obitelji su izuzetna supstratna nespecifičnost te tipični GDSL motiv oko serina iz katalitičke trijade, koji se razlikuje od GXSXG motiva iz većine dosad istraženih lipaza. TesA sudjeluje u putu sinteze masnih kiselina te katalizira hidrolizu čitavog niza supstrata (estera i tioestera različite duljine lanca masnih kiselina). U ovom radu pokušali smo razjasniti molekularnu osnovu supstratne specifičnosti i supstratnog promiskuiteta enzima TesA korištenjem računalnih metoda temeljenih na polju sila, prvenstveno molekularne dinamike (MD). Rezultati simulacija poduprti su eksperimentalnim mjerenjem aktivnosti enzima prema različitim supstratima. Simulacijama smo objasnili smještanje supstrata različitih veličina i oblika u aktivno mjesto TesA te promjene u strukturi i dinamici aktivnog mjesta izazvanih prisutnošću različitih supstrata te uvođenjem mutacija. Utjecaj izabranih mutacija istražen je in silico i eksperimentalnim metodama.

Abstract (english)

Acyl-­CoA thioesterase I (TesA) from the bacterium Pseudomonas aeruginosa is a lipolytic enzyme that belongs to the family of GDSL hydrolases. The GDSL hydrolase family is a relatively new and not so well investigated group of enzymes with a great potential for application in pharmaceutical and food industry. Members of this enzyme family have typical GDSL motif surrounding the serine form the catalytic triad, which is different from the GXSXG motif found in other lipases. Some members of the GDSL family show large substrate promiscuity. Enzyme TesA is engaged in the fatty acid synthesis, and it hydrolyses a large number of different substrates (esters and thioesters with different fatty acid chain length). In this thesis we have tried to determine the molecular basis of substrate specificity and promiscuity of enzyme TesA using force field based computational methods, primarily molecular dynamics (MD) simulations. Simulation results are supported by experimentally determined enzyme activity towards different substrates. We have investigated the positioning of differently sized and shaped substrates in the binding site of the enzyme TesA, as well as the changes in structure and dynamics of the binding site caused by the presence of different substrates. According to the simulation results, mutations that should affect TesA activity were proposed. The proposed mutants were investigated by in silico and experimental methods.

• Keywords: molekularna dinamika; supstratna specifičnost; in silico mutacije
• Keywords (english): molecular dynamics; substrate specificity; in silico mutations
• Language: croatian
• URN:NBN: urn:nbn:hr:217:468247
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-04-27 12:29:23