Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana

Research output: Contribution to journalArticleResearchpeer review

Authors

Research Organisations

View graph of relations

Details

Original languageEnglish
Pages (from-to)140-149
Number of pages10
JournalPhysiologia plantarum
Volume135
Issue number2
Publication statusPublished - 1 Feb 2009

Abstract

Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana, three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Physiology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Genetics
  • Agricultural and Biological Sciences(all)
  • Plant Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Cell Biology

Cite this

Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana. / Klein, Marion; Papenbrock, Jutta.
In: Physiologia plantarum, Vol. 135, No. 2, 01.02.2009, p. 140-149.

Research output: Contribution to journalArticleResearchpeer review

Download
@article{135497df864f480cb0adbcec47ac7953,
title = "Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana",
abstract = "Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana, three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.",
author = "Marion Klein and Jutta Papenbrock",
year = "2009",
month = feb,
day = "1",
doi = "10.1111/j.1399-3054.2008.01182.x",
language = "English",
volume = "135",
pages = "140--149",
journal = "Physiologia plantarum",
issn = "0031-9317",
publisher = "Wiley-Blackwell Publishing Ltd",
number = "2",

}

Download

TY - JOUR

T1 - Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana

AU - Klein, Marion

AU - Papenbrock, Jutta

PY - 2009/2/1

Y1 - 2009/2/1

N2 - Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana, three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.

AB - Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana, three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.

UR - http://www.scopus.com/inward/record.url?scp=58249105842&partnerID=8YFLogxK

U2 - 10.1111/j.1399-3054.2008.01182.x

DO - 10.1111/j.1399-3054.2008.01182.x

M3 - Article

C2 - 19077143

AN - SCOPUS:58249105842

VL - 135

SP - 140

EP - 149

JO - Physiologia plantarum

JF - Physiologia plantarum

SN - 0031-9317

IS - 2

ER -