Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases

master's thesis

• Title (croatian): Istraživanje reakcijskih mehanizama kratkolančanih dehidrogenaza/reduktaza epimeraza i dekarboksilaza
• Author: Fran Debanić
• Mentor: Anita Slavica (mentor)
• Committee member: Mirela Ivančić Šantek (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: 2021-07-23, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

UDP-GlcA methyl ester, a substrate analogue for short-chain dehydrogenase/reductase epimerases and decarboxylases - human UDP-Xylose synthase (UXS), UDP-Apiose/Xylose synthase (UAXS) and UDP-glucuronic acid 4-epimerase (UGAepi), was successfully synthesized and purified and thus enabled investigation of their enzymatic mechanisms. The three enzymes were expressed in E. coli and purified to homogeneity. Both decarboxylases (UXS and UAXS) converted UDP-GlcA methyl ester (0.71 U/mg and 0.03 U/mg, respectively) in a comparable manner to conversion of their natural substrate - UDP-glucuronic acid (UDP-GlcA, 1.6 U/mg and 0.15 U/mg, respectively). Newly established methanol assay proved that the two decarboxylases hydrolyze the ester before the decaborxylation reaction while UGAepi does not perform the hydrolysis. UGAepi epimerizes faster the substrate analogue (0.65 U/mg) than the natural substrate (0.53 U/mg). Additionally, gene encoding UAXS from Geminococcus roseus was successfully expressed in E. coli and resulting enzyme (GrUAXS) posses’ lower activity towards UDP-GlcA (2.1 mU/mg) than it's mutant (S120C; 12.3 mU/mg) supporting the importance of cysteine residue in the active site of the enzyme.

Abstract (croatian)

UDP-GlcA metil ester, analog supstrata za kratkolančane dehidrogenaze/redukataze epimeraze i dekarboksilaze - humanu UDP-ksiloza sintazu (UXS), UDP-apioza/ksiloza sintazu (UAXS) i UDP-glukuronska kiselina 4-epimerazu (UGAepi), uspješno je sintetiziran i pročišćen, čime je omogućeno istraživanje enzimskih mehanizama ovih katalizatora. Tri enzima su eksprimirana u stanicama E. coli i pročišćena. Obje dekarboksilaze (UXS and UAXS) su konvertirale UDP-GlcA metil ester (redom 0.71 U/mg i 0.03 U/mg) slično konverziji njihova prirodnog supstrata - UDP- glukuronske kiseline (UDP-GlcA, redom 1.6 U/mg i 0.15 U/mg). Pomoću novoustanovljene metode za određivanje metanola utvrđeno je da dvije dekarboksilaze hidroliziraju ester prije reakcije dekarboksilacije, dok UGAepi ne provodi hidrolizu estera. UGAepi brže epimerizira analog supstrata (0.65 U/mg) nego svoj prirodni supstrat (0.53 U/mg). Dodatno, gen koji kodira UAXS iz Geminococcus roseus je uspješno eksprimiran u E. coli i dobiveni enzim (GrUAXS) ima nižu aktivnost prema UDP-GlcA (2.1 mU/mg) nego njegov mutant (S120C, 12.3 mU/mg), što podupire važnu ulogu cisteina u aktivnom mjestu ovoga enzima

• Keywords: GrUAXS; UAXS; UDP-glucuronic acid methyl ester; UGAepi; UXS
• Keywords (english): GrUAXS; UAXS; UDP-glukuronksa kiselina metil ester; UGAepi; UXS
• Language: english
• URN:NBN: urn:nbn:hr:159:077009
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-10-05 10:41:08