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Sibrian, Carlos, 2009. Crystallization attempt of TGG5 and mutant studies of TGG4 myrosinase from thale cress (Arabidopsis thaliana). Second cycle, A2E. Uppsala: SLU, Dept. of Plant Biology and Forest Genetics (until 131231)

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Abstract

Myrosinase is a glucosidase normally found in plants of the order Capparales and that is involved in the breakdown of glucosinolates releasing toxic breakdown products. Together myrosinase and glucosinolates form a plant defence system that deters pests and herbivores by a barrage of toxic breakdown products. Many of the crops containing the myrosinase-glucosinolate system are valuable vegetable- or oil-crops making studies on myrosinase very important.

TGG5 and TGG4 are myrosinases belonging to Arabidopsis thaliana. The advantages with using myrosinase from a model organism is the extensive knowledge of the organism and the many tools and methods developed. Crystallization attempt were performed with recombinant TGG5 (containing a his-tag) where the myrosinase was expressed in Pichia pastoris and purified using immobilized metal-ion affinity chromatography (IMAC) and either cation-exchange chromatography or gel filtration chromatography. No crystals were observed but it seemed like the LiSO4 conferred stability to the myrosinase enzyme maybe due to interaction with the sulphate binding site of the enzymes usually used for binding the sulphate containing substrate. TGG4 is another myrosinase isozyme found in A. thaliana and is very similar to TGG5 sharing 97% sequence identity. TGG4 mutants were designed using the structure of a crystallised myrosinase from Sinapsis alba as reference. Mutants were modified with respect to active site and residues predicted to be involved in substrate binding and co-factor (ascorbate) binding. Some of the findings were the reduced catalytic activity encountered when mutating the glutamine residue (replaced by an glutamate residue in O-β-glucosidases) and that converting the glutamine back to glutamate did not confer any additional catalytic activity.

Main title:Crystallization attempt of TGG5 and mutant studies of TGG4 myrosinase from thale cress (Arabidopsis thaliana)
Authors:Sibrian, Carlos
Supervisor:Meijer, Johan
Examiner:Sitbon, Folke
Series:Examensarbete / Sveriges lantbruksuniversitet, Institutionen för växtbiologi och skogsgenetik
Volume/Sequential designation:105
Year of Publication:2009
Level and depth descriptor:Second cycle, A2E
Department:(NL, NJ) > Dept. of Plant Biology and Forest Genetics (until 131231)
Keywords:Myrosinase, glucosinolate, Arabidopsis thaliana, mutant studies, TGG4
URN:NBN:urn:nbn:se:slu:epsilon-8-153
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-8-153
Subjects:Pests of plants
Plant physiology and biochemistry
Weeds and weed control
Language:English
Deposited On:01 Sep 2009 10:41
Metadata Last Modified:20 Apr 2012 14:09

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