Molecular and biochemical characterization of two brassinosteroid sulfotransferases from Arabidopsis, AtST4a (At2g14920) and AtST1 (At2g03760)

Abstract

Mammalian sulfotransferases (EC 2.8.2) are involved in many important facets of steroid hormone activity and metabolism. In this study, Arabidopsis AtST4a and AtST1 were identified and characterized as brassinosteroid sulfotransferases that appear to be involved in different aspects of hormone regulation. The two proteins share 44% identity in amino acid sequence, and belong to different plant sulfotransferase families. AtST4a was specific for biologically active end products of the brassinosteroid pathway. The enzyme sulfated brassinosteroids with diverse side-chain structures, including 24-epibrassinosteroids and the naturally occurring (22R, 23R)-28-homobrassinosteroids. AtST4a belongs to a small subfamily of sulfotransferases having two other members, AtST4b and -c. Among the three recombinant enzymes, only AtST4a was catalytically active with brassinosteroids. Transcript expression of AtST4 subfamily members was largely specific to the root. AtST4b- and -c transcript levels were induced by treatment with trans-zeatin, while AtST4a was repressed under the same conditions, supporting a divergent function of AtST4a. Co-regulation of AtST4b and -c correlated with their location in tandem on chromosome 1. AtST1 was stereospecific for 24-epibrassinosteroids, with a substrate preference for the metabolic precursor 24-epicathasterone, and exhibited catalytic activity with hydroxysteroids and estrogens. To gain more insight into this dual activity with plant and mammalian steroids, enzymatic activities of human steroid sulfotransferases toward brassinosteroids were characterized. The dehydroepiandrosterone sulfotransferase SULT2A1 displayed catalytic activity with a selected set of 24-epibrassinolide precursors, including 24-epicathasterone, with specific activities comparable to that measured for the endogenous substrate dehydroepiandrosterone. The comparable activity profiles of AtST1 and SULT2A1 suggest a similar architecture of the acceptor-binding site between the two enzymes, and may potentially reflect a common ability to conjugate certain xenobiotics.

Salicylic Acid Biosynthesis and Metabolism

“…Characterization of SOT12 revealed that it catalyzes the transfer of a sulphuryl group ) to the 2-OH position of SA in vitro; thus, Baek et al (2010) postulated that this protein functions as an SA sulfotransferase in planta. However, sulphonated SA was not detected in planta and sulphonated SA formation in vitro required high concentrations of the substrate (K m = 0.44 mM for SA (Baek et al, 2010) compared with K m = 7 mM for the brassinosteroid 24-epicathasterone (Marsolais et al, 2007)). Although the K m of SOT12 for SA is in the range of the SA glucosyl transferases, it remains to be established if SOT12 acts on SA in vivo or primarily sulfonates another target.…”

The Arabidopsis geneATST4ais not a typical brassinosteroids catabolic gene

“…20 Not enough genetic data are available, however, to support this role of ATST4a in BR catabolism in Arabidopsis. This study uses both gain-of-function and lossof-function genetic approaches to further analyze the role of ATST4a in Arabidopsis.…”

“…6 The latter is unlikely since biochemical analysis shows that the closest homologs of ATST4a (ATST4b and ATST4c) do not display any affinity for brassinosteroids. 20 In vitro biochemical analysis can give important clues to the function of an enzyme. However enzyme activity and function at a tissue or cellular level may be dependent on certain in vivo conditions and therefore difficult to assess.…”

“…16 ATST4a was cloned on the basis of homology to ATST1. 20 ATST1 is an ortholog of BNST (steroid sulfotransferase from Brassica napus) proteins and displays similar catalytic activities and substrate specificity.…”

“…19 In vitro protein expression and sulfotransferase assay studies demonstrate that ATST4a has catalytic activity with BRs. 20 Catalytic activity of ATST4a is specific for biologically active end products of the BR biosynthetic pathway, including castasterone, brassinolide, related 24-epimers, and the naturally occurring (22R, 23R)-28-homobrassinosteroids. The addition of a sulfate group to the steroid molecules may cause a change in their activity and therefore play a role in steroid homeostasis.…”