Synthesis of mechanistic probes and mutational analysis of the sugar C4/C6 interactions in cytidine 5’-diphosphate-tyvelose 2-epimerase from Thermodesulfatator atlanticus

master's thesis

• Title (croatian): Sinteza mehanističkih proba i mutacijska analiza C4/C6 interakcija ugljikohidrata i citidin 5'-difosfat tiveloze 2-epimeraze iz Thermodesulfatator atlanticus
• Author: Magdalena Merkaš
• Mentor: Anita Slavica (mentor)
• Committee member: Renata Teparić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2021-07-27, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Mechanistic probes for investigation of CDP-Tyvelose 2-Epimerase - like enzyme from Thermodesulfatator atlanticus (TaCPa2E) activity were synthesized. CDP-Lyxose, the mechanistic probe for characterization of C6 interaction with TaCPa2E, was obtained by using chemical approach while CDP-4-Fluoro-4-deoxy-D-Glucose, the mechanistic probe for studying C4 interaction with the enzyme, was yielded by using enzymatic anomeric approach. The later substrate was produced after galactokinase from Streptococcus pneumoniae (GalKSpe4) and UDP-glucose pyrophosporylase from Bifidobacterium longum (UGPase), were produced in E. coli, purified, and therefrom used in the manufacturing of the substrate. Active mutants of TaCPa2E - G206A and D204E were created and used in characterization of the C4/C6 interactions. The highest specific activity towards CDP-Lyxose was determined for wild-type TaCPa2E (0.27 U/ml) and for the two mutants - G206A (0.50 U/ml) and D204E (0.41 U/ml), while CDP-Mannose seems to be better accepted by all three variants (0.02 U/ml for all three forms of the enzyme) than CDP-Glucose. TaCPa2E showed more affinity towards C5 CDP-activated substrate than towards CDP-activated hexoses. Further research might shed more light on CDP-Glucose - and CDP-4-Fluoro-4-deoxy-D-Glucose - enzyme interactions.

Abstract (croatian)

Sintetizirane su mehnističke probe za istraživanje aktivnosti enzima sličnog CDP-Tiveloza 2-Epimerazi iz Thermodesulfatator atlanticus (TaCPa2E). CDP-liksoza, mehanistička proba za karakterizaciju C6 interakcije supstrata s TaCPa2E je dobivena kemijskom sintezom, dok je CDP-4-Fluoro-4-deoksi-D-glukoza, mehanistička proba za istraživanje C4 interakcije supstrata s enzimom, dobivena enzimskom sintezom. Ovaj drugi supstrat je proizveden nakon što su galaktokinaza iz Streptococcus pneumoniae (GalKSpe4) i UDP-glukoza pirofosforilaza iz Bifidobacterium longum (UGPase) sintetizirane u E. coli, pročišćene i onda korištene u proizvodnji ovoga supstrata. Aktivni mutanti TaCPa2E - G206A i D204E također su sintetizirani i korišteni za karakterizaciju C4/C6 interakcija. Najveću specifičnu aktivnost prema CDP-liksozi pokazali su divlji tip TaCPa2E (0.27 U/ml) i dva mutanta - G206A (0.50 U/ml) i D204E (0.41 U/ml), dok je CDP-manoza bolje prihvaćena kao susptrat kod sva tri enzima (0.02 U/ml za sve tri forme ovoga enzima) od CDP-glukoze. Ustanovljeno je da TaCPa2E pokazuje bolju aktivnost prema C5 CDP-aktiviranom supstratu nego prema CDP-aktiviranim heksozama. Potrebna su daljnja istraživanja kako bi se bolje pojasnile interakcije CDP-glukoze i CDP-4-Fluoro-4-deoksi-D-glukoze s ovim enzimom.

• Keywords: CDP-Tyvelose 2-Epimerase-like enzyme; mechanistic probes; mutational analysis
• Keywords (english): CDP-Tiveloza 2-Epimerazi-sličan enzim; mehanističke probe; mutacijska analiza
• Language: english
• URN:NBN: urn:nbn:hr:159:652273
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-10-05 11:50:32