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Mutagenesis of the sugar donor site of the Arabidopsis thaliana glycosyltransferase UGT72B1
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
2010 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

The Arabidopsis thaliana glycosyltransferase UGT72B1 is one of many enzymes which catalyze the reaction oflinking a glucose moiety from UDP-glucose to an acceptor molecule, in this case a chloroaniline or a chlorophenol. This is part of a detoxification system of the plant cell, similar to that in humans where a glucuronosyltransferases are enabling drug metabolism. It would be of interest to investigate the activity of the human enzyme towards different pharmaceuticals and determine the effect the linkage of glucose has to properties of the compounds. However, the human enzymes are membrane proteins and thus difficult to purify and crystallize. Here, an attempt was made to instead change the substrate specificity of UGT72B1 from UDPglucose to UDP-glucuronic acid. Combination of the four point mutations G18S, P139R, W367S and AG387ED were introduced in UGT72B1. However, no UDP-glucuronic acid activity was obtained. Single mutants W367S and AG387ED retained similar activity as of the wildtype while P139R had highly reduced activity and G18S was not expressed at all. All other combinations of mutations resulted in even less activity. Four chimeric proteins were also constructed. They were combinations of the UGT72B1 and the human enzyme UGT2B4. These were all soluble proteins but no activity could be determined.

Abstract [sv]

Glykosyltransferaset UGT72B1 från Arabidopsis thaliana är ett av många enzymer som katalyserar reaktionen där en glukosenhet från UDP-glukos länkas till en acceptormolekyl, i det här fallet en kloranilin eller en klorfenol. Det är en del av ett detoxifieringssytem i växtcellen, som liknar det i människan, där ett glukuronosyltransferas möjliggör nedbrytning av bl.a. läkemedel. Det vore intressant att kunna undersöka de humana enzymernas aktivitet mot olika läkemedel och även fastställa effekten glukoslänkningen har på dessa substansers egenskaper. De humana enzymerna är dock membranprotein och är därför svåra att rena fram och att kristallisera. Här har istället ett försök gjorts för att ändra substratspecificiteten hos UGT72B1 från UDP-glukos till UDP-glukuronsyra. Kombinationer av de fyra punktmutationerna G18S, P139R, W367S och AG387ED introducerades i UGT72B1. Ingen aktivitet med UDP-glukuronsyra erhölls dock. Enkelmutanterna W367S och AG387ED bibehöll liknande aktivitet som vildtypen, medan P139R hade starkt reducerad aktivitet och G18S uttrycktes inte alls. Alla andra kombinationer av mutationer resulterade i ännu lägre aktivitet. Fyra chimeriska proteiner konstruerades också. De skapades genom kombination av UGT72B1 och det humana enzymet UGT2B4. Dessa var alla lösliga proteiner men ingen av dem uppvisade någon aktivitet.

Place, publisher, year, edition, pages
2010. , 55 p.
Keyword [en]
UGT72B1 UGT2B4 glycosyltransferase substrate specificity UDP-glucose UDP-glucuronic acid activity SOE PCR quik change protein expression protein purification HPLC
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:liu:diva-66483ISRN: LITH-IFM-EX--10/2278--SEOAI: diva2:404508
2010-09-14, Linköping, 13:00 (Swedish)
Physics, Chemistry, Mathematics
Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2011-03-17Bibliographically approved

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