Strukturna karakterizacija Nudix hidrolaze iz amebe Dictyostelium discoideum

master's thesis

• Title (english): Structural characterization of Nudix hydrolase from the amoeba Dictyostelium discoideum
• Author: Ana Vlašić
• Mentor: Marija Luić (mentor)
• Committee member: Dubravka Matković-Čalogović (predsjednik povjerenstva)
• Committee member: Marija Luić (član povjerenstva)
• Committee member: Sanda Rončević (član povjerenstva)
• Committee member: Biserka Prugovečki (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2018-09-18, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Istraživanje biljnih dipeptidil-peptidaza III (DPP III) rezultiralo je neočekivanom spoznajom da postoji enzim s dvije funkcionalne domene (Nudix i DPP III), koji usto pokazuje i atipičnu aktivnost Nudix hidrolaza prema supstratu izopentenil-difosfatu (IPP). U svrhu strukturne karakterizacije Nudix domene ovog interesantnog enzima, a na temelju značajne sličnosti primarnih struktura, za istraživanje je odabrana Nudix hidrolaza iz amebe Dictyostelium discoideum (DdNudix). Nudix hidrolaze pripadaju nadporodici hidrolitičkih Mg2+-ovisnih enzima koji djeluju kao fosfataze ili pirofosfohidrolaze.
Divlji tip i neaktivni mutant E82A DdNudix, obilježeni histidinskim privjescima, prekomjerno su eksprimirani u bakteriji Escherichia coli, te pročišćeni afinitetnom kromatografijom i gel filtracijom. Optimizirani su uvjeti kristalizacije proteina DdNudix. Metodom anomalnog raspršenja riješena je struktura divljeg tipa proteina obilježenog selenometioninom do razlučivanja od 2,4 Å. Metodom molekulske zamjene riješena je struktura mutanta proteina E82A sa supstratom IPP do razlučivanja od 1,6 Å. Detaljnom strukturnom analizom ustanovljene su sličnosti i razlike s Nudix hidrolazama kao i s izopentenil-difosfat-izomerazama.

Abstract (english)

The investigation of plant dipeptidyl peptidase III (DPP III) lead to an unexpected discovery, the existence of an enzyme with dual function (Nudix and DPP III), which also shows the atypic Nudix hydrolase activity against the isopentenyl diphosphate (IPP) substrate. For the purpose of structural characterization of the Nudix domain of this interesting enzyme, and based on the significant similarity of primary structures, Nudix hydrolase from amoeba Dictyostelium discoideum (DdNudix) was selected for this study. Nudix hydrolases are members of superfamily of hydrolytic Mg2+-dependent enzymes acting as phosphatases or pyrophosphohydrates.
The wild type and inactive E82A mutant DdNudix, labeled with histidine tags, were over-expressed in Escherichia coli bacteria and purified by affinity chromatography and gel filtration. The conditions for crystallization of DdNudix protein were optimized. Using anomalous scattering, the structure of the selenomethionine labeled wild-type protein was resolved up to the resolution of 2.4 Å. Structure of the protein mutant, E82A, in complex with IPP substrate was solved with the molecular replacement method up to the resolution of 1.6 Å. Detailed structural analysis revealed similarities and differences of Nudix hydrolases as well as isopentenyl diphosphate isomerases.

• Keywords: Nudix hidrolaze; izopentenil-difosfat-izomeraze; Dictyostelium discoideum; kristalna struktura; metoda SAD; metoda molekulske zamjene
• Keywords (english): Nudix hydrolase; isopentenyl diphosphate isomerase; Dictyostelium discoideum; crystal structure; SAD method; molecular replacement method
• Language: croatian
• URN:NBN: urn:nbn:hr:217:322098
• 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: 2020-09-18
• Created on: 2018-11-22 13:08:29