Details
| Original language | English |
|---|---|
| Pages (from-to) | 122-136 |
| Number of pages | 15 |
| Journal | FEBS Journal |
| Volume | 273 |
| Issue number | 1 |
| Publication status | Published - 1 Jan 2006 |
Abstract
Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3′-phosphoadenosine 5′-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light/darkness cycle. High (500 μm) and low (50 μm) sulfate concentrations in the medium did not influence the levels of expression.
Keywords
- 3′-phosphoadenosine 5′-phosphosulfate (PAPS), Glucosinolates, Subcellular localization, Substrate specificity, Sulfotransferase
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: FEBS Journal, Vol. 273, No. 1, 01.01.2006, p. 122-136.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - The three desulfoglucosinolate sulfotransferase proteins in Arabidopsis have different substrate specificities and are differentially expressed
AU - Klein, Marion
AU - Reichelt, Michael
AU - Gershenzon, Jonathan
AU - Papenbrock, Jutta
PY - 2006/1/1
Y1 - 2006/1/1
N2 - Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3′-phosphoadenosine 5′-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light/darkness cycle. High (500 μm) and low (50 μm) sulfate concentrations in the medium did not influence the levels of expression.
AB - Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3′-phosphoadenosine 5′-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light/darkness cycle. High (500 μm) and low (50 μm) sulfate concentrations in the medium did not influence the levels of expression.
KW - 3′-phosphoadenosine 5′-phosphosulfate (PAPS)
KW - Glucosinolates
KW - Subcellular localization
KW - Substrate specificity
KW - Sulfotransferase
UR - http://www.scopus.com/inward/record.url?scp=33644875639&partnerID=8YFLogxK
U2 - 10.1111/j.1742-4658.2005.05048.x
DO - 10.1111/j.1742-4658.2005.05048.x
M3 - Article
C2 - 16367753
AN - SCOPUS:33644875639
VL - 273
SP - 122
EP - 136
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 1
ER -