Strukturna istraživanja enzima purinske nukleozidne fosforilaze iz bakterije Helicobacter pylori

master's thesis

• Title (english): Structural investigation of enzyme purine nucleoside phosphorylase from bacteria Helicobacter pylori
• Author: Lucija Papa
• Mentor: Marija Luić (mentor)
• Committee member: Dubravka Matković-Čalogović (predsjednik povjerenstva)
• Committee member: Marija Luić (član povjerenstva)
• Committee member: Davor Kovačević (član povjerenstva)
• Committee member: Marijana Đaković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2017-02-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Bakterija Helicobacter pylori, patogen ljudskog probavnog sustava, za biosintezu purinskih nukleozida koristi samo pomodni metabolički put. Smatra se da bi inhibicija ključnih enzima navedenog puta mogla inhibirati rast ove bakterije. Jedan od takvih enzima je purinska nukleozidna fosforilaza (PNP), koja katalizira fosforolitičko cijepanje glikozidne veze u purinskim nukleozidima.
Kako bi se što bolje razumjelo vezanje liganada u aktivno mjesto PNP, pripremljeni su kristali binarnih kompleksa PNP s fosfatnim anionom, te s formicinom A - strukturnim analogom purinskog nukleozida adenozina, kao i kristali ternarnog kompleksa koji sadrži oba liganda. Trodimenzijske strukture kompleksa određene su metodom difrakcije rendgenskog zračenja na jediničnom kristalu. Vezanje fosfatnih aniona u aktivna mjesta enzima, koje je mogude samo u uvjetima snižene ionske jakosti, vjerojatno dovodi do konformacijske promjene u podjedinici i zatvaranja aktivnog mjesta. U otvorenim aktivnim mjestima konformacija formicina A razlikuje se od one u zatvorenim aktivnim mjestima. Navedeno je u skladu s trenutno pretpostavljenim katalitičkim mehanizmom za sličan PNP iz bakterije E. coli.

Abstract (english)

Bacterium Helicobacter pylori is a known human digestive system pathogen. For biosynthesis of purine nucleosides it relies exclusively on salvage pathway, therefore, new medicine could be developed by inhibiting salvage pathway's crucial enzymes. Purine nucleoside phosphorylase (PNP), one of those enzymes, catalyzes phosphorolytic cleavage of the glycosidic bond in purine nucleosides.
Binary complexes of PNP with phosphate ions (natural substrate) and formycin A (natural substrate's uncleavable structural analogue), and ternary complex of PNP with both of those ligands were prepared and crystallized in order to explore ligand binding into the active sites of the enzyme. Structures of the prepared complexes were determined using single-crystal X-ray crystallography. Closing of the active sites is probably caused by conformational changes driven by phosphate binding, possible only in low ionic strength conditions. Formycin A adopts different conformations in open and closed active sites. Observed effects are in agreement with the currently proposed catalytic mechanism of the similar PNP protein from E. coli.

• Keywords: difrakcija rendgenskog zračenja na jediničnom kristalu; formicin A; fosfat; Helicobacter pylori; proteinska kristalografija; purinska nukleozidna fosforilaza
• Keywords (english): formycin A; Helicobacter pylori; phosphate; protein crystallography; purine nucleoside phosphorylase; single-crystal X-ray crystallography
• Language: croatian
• URN:NBN: urn:nbn:hr:217:510076
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2018-08-21
• Created on: 2017-03-03 13:25:22