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.

The fusion of genomes leads to more options: A comparative investigation on the desulfo-glucosinolate sulfotransferases of Brassica napus and homologous proteins of Arabidopsis thaliana

“…BnSOT17-a preferred long-chained methionine-derived ds-Gls 4MTB and 5MTP. These results correspond with the substrate specificities to the AtSOT homologs (Klein and Papenbrock, 2009). Therefore, knowledge about AtSOT16 and AtSOT17 could generally be transferred to Brassica species.…”

“…The preferences of AtSOT17 and 18 are long chained aliphatic methylthio Gls, such as ds-7-methylthioheptyl Gl and ds-8-methylthiooctyl Gl (Klein et al, 2006;Klein and Papenbrock, 2009;Piotrowski et al, 2004). Detailed analysis of AtSOT18 from different ecotypes showed that the exchange of only one or two amino acids can lead to different affinities to the substrate and PAPS (Klein et al, 2006;Klein and Papenbrock, 2009;Luczak et al, 2013). The questions remain, whether AtSOT16 to 18 are specific for A. thaliana, or if there are homologous SOTs in other Glsynthesizing plants.…”

“…These substrates were chosen for a better comparison, since the respective AtSOT values were determined previously (Klein and Papenbrock, 2009;Luczak et al, 2013;Piotrowski et al, 2004). The determined K m values ranged from 90 ± 43 mM to 281 ± 193 mM, while the V max values ranged from 58 ± 18 pkat mg À1 to 638 ± 291 pkat mg À1 (Table 3).…”

“…Characterization of AtSOT16 to 18 revealed that AtSOT16 prefers indolic ds-Gls, such as indol-3-yl-methyl Gl (I3M), as substrate, followed by aliphatic ds-Gls, such as ds-3-methylthiopropyl Gl (3MTP) and ds-4-methylthiobutyl Gl (4MTB). The preferences of AtSOT17 and 18 are long chained aliphatic methylthio Gls, such as ds-7-methylthioheptyl Gl and ds-8-methylthiooctyl Gl (Klein et al, 2006;Klein and Papenbrock, 2009;Piotrowski et al, 2004). Detailed analysis of AtSOT18 from different ecotypes showed that the exchange of only one or two amino acids can lead to different affinities to the substrate and PAPS (Klein et al, 2006;Klein and Papenbrock, 2009;Luczak et al, 2013).…”

“…For ten of those the used substrates have been determined (Hirschmann et al, 2014). Eighteen SOTs (AtSOT1 -18) were previously divided in seven groups and share about 51% sequence identity on amino acid level (Klein and Papenbrock, 2009). Except for AtSOT3, 4 and 10, none of those contain introns.…”