Biokemijska karakterizacija adenilosukcinat-sintetaze bakterije Helicobacter pylori eksperimentalnim i računalnim metodama

doctoral thesis

• Title (english): Biochemical characterization of adenylosuccinate synthetase from Helicobacter pylori using experimental and computational methods
• Author: Ante Bubić
• Mentor: Branimir Bertoša (mentor)
• Mentor: Ivana Leščić Ašler (komentor)
• Committee member: Jasmina Rokov Plavec (predsjednik povjerenstva)
• Committee member: Biserka Prugovečki (član povjerenstva)
• Committee member: Sanja Tomić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2021, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Adenilosukcinat-sintetaza (AdSS) je esencijalan enzim u biosintezi purina, i de novo, i reciklirajućim putem. U ovoj disertaciji je računalno i eksperimentalno karakterizirana AdSS iz bakterije Helicobacter pylori koja pripada skupini I kancerogena. Pročišćen je enzim sa i bez histidinskog privjeska na C-kraju. Za oba enzima su određeni specifična aktivnost, temperaturno i pH područje stabilnosti, te kinetički parametri prema supstratima i inhibitorima. Dobiveni rezultati ukazuju na to da dodani histidinski privjesak nema značajan utjecaj na funkciju enzima. Riješena kristalna struktura AdSS pri visokoj rezoluciji, korištena za karakterizaciju enzima računalnim metodama, otkriva stvaranje stabilnog međuprodukta 6'-fosforil-IMP u aktivnom mjestu enzima. Računalnim metodama identificirane su ključne interakcije za stabilizaciju supstrata u aktivnom mjestu. Analiza kristalne strukture i računalna karakterizacija interakcija proteina s ligandima ukazuju na veliku sličnost AdSS iz bakterije H. pylori s AdSS iz vrste Escherichia coli, stoga se može pretpostaviti i sličan mehanizam rada enzima.

Abstract (english)

Adenylosuccinate synthetase (AdSS) is an essential enzyme in de novo and recycling pathway of purine biosynthesis. In this thesis, AdSS from bacterium Helicobacter pylori, a group I carcinogen, was characterized experimentally and computationally. Enzyme with and without histidine tag at the C-terminus was purified. Specific activity, temperature and pH stability range, and kinetic parameters toward substrates and inhibitors were determined for both enzymes. Obtained results indicate that the added histidine tag has no significant effect on enzyme function. Solved crystal structure at high resolution, used to characterize the enzyme computationally, reveals the formation of a stable intermediate 6'-phosphoryl-IMP in the active site. Computational methods were used to identify key interactions for stabilisation of substrates in the active site. Analysis of crystal structure and computational characterization of protein-ligand interactions indicate high similarity of H. pylori AdSS to Escherichia coli AdSS, therefore the similar molecular mechanism for this enzyme can be assumed.

• Keywords: adenilosukcinat-sintetaza; enzimska kinetika; Helicobacter pylori; histidinski privjesak; kristalna struktura; molekularna dinamika
• Keywords (english): adenylosuccinate synthetase; crystal structure; enzyme kinetics; Helicobacter pylori; histidine tag; molecular dynamics
• Language: croatian
• URN:NBN: urn:nbn:hr:217:625124
• Promotion: 2021
• Project: Number: IP-2013-11-7423; Title: Enzimi purinskog reciklirajućeg ciklusa iz Helicobacter pylori i Escherichie coli; Title: Purine salvage pathway enzymes from Helicobacter pylori and Escherichia coli; Acronym: PSPE; Leader: Marija Luić; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Project: Number: IP-2019-04-6764; Title: Alosterički komunikacijski putevi u oligomernim enzimima; Title: Allosteric communication pathways in oligomeric enzymes; Acronym: ALOKOMP/ALOCOMP; Leader: Zoran Štefanić; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Study programme: Title: Doctoral study in chemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija (doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija)
• Type of resource: Text
• Extent: 95 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-09-27 11:58:08