Crystallization attempt of TGG5 and mutant studies of TGG4 myrosinase from thale cress (Arabidopsis thaliana)
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...
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| Formato: | H2 |
| Lenguaje: | Inglés |
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SLU/Dept. of Plant Biology and Forest Genetics (until 131231)
2009
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| Materias: |
| _version_ | 1855570192377053184 |
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| author | Sibrian, Carlos |
| author_browse | Sibrian, Carlos |
| author_facet | Sibrian, Carlos |
| author_sort | Sibrian, Carlos |
| collection | Epsilon Archive for Student Projects |
| description | 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.
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| format | H2 |
| id | RepoSLU464 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés |
| publishDate | 2009 |
| publishDateSort | 2009 |
| publisher | SLU/Dept. of Plant Biology and Forest Genetics (until 131231) |
| publisherStr | SLU/Dept. of Plant Biology and Forest Genetics (until 131231) |
| record_format | eprints |
| spelling | RepoSLU4642012-04-20T14:09:44Z Crystallization attempt of TGG5 and mutant studies of TGG4 myrosinase from thale cress (Arabidopsis thaliana) Kristalliserings försök med TGG5 och mutant studier av TGG4 myrosinase från backtrav (Arabidopsis thaliana) Sibrian, Carlos Myrosinase glucosinolate Arabidopsis thaliana mutant studies TGG4 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. SLU/Dept. of Plant Biology and Forest Genetics (until 131231) 2009 H2 eng https://stud.epsilon.slu.se/464/ |
| spellingShingle | Myrosinase glucosinolate Arabidopsis thaliana mutant studies TGG4 Sibrian, Carlos Crystallization attempt of TGG5 and mutant studies of TGG4 myrosinase from thale cress (Arabidopsis thaliana) |
| title | Crystallization attempt of TGG5 and
mutant studies of TGG4 myrosinase from
thale cress (Arabidopsis thaliana) |
| title_full | Crystallization attempt of TGG5 and
mutant studies of TGG4 myrosinase from
thale cress (Arabidopsis thaliana) |
| title_fullStr | Crystallization attempt of TGG5 and
mutant studies of TGG4 myrosinase from
thale cress (Arabidopsis thaliana) |
| title_full_unstemmed | Crystallization attempt of TGG5 and
mutant studies of TGG4 myrosinase from
thale cress (Arabidopsis thaliana) |
| title_short | Crystallization attempt of TGG5 and
mutant studies of TGG4 myrosinase from
thale cress (Arabidopsis thaliana) |
| title_sort | crystallization attempt of tgg5 and
mutant studies of tgg4 myrosinase from
thale cress (arabidopsis thaliana) |
| topic | Myrosinase glucosinolate Arabidopsis thaliana mutant studies TGG4 |