Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana

Abstract

The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme preferred flavonoid glycosides (e.g. kaempferol-3-glucoside and quercetin-3-glucoside) rather than their aglycone counterparts. Among a series of hydroxyflavones tested, AtSULT202B7 showed the enzymatic activity only for 7-hydroxyflavone. pH-dependency study showed that the optimum pH was relatively low (pH 5.5) compared with those (pH 6.0-8.5) previously reported for other isoforms. Based on the comparison of high performance (pressure) liquid chromatography (HPLC) retention times between sulfated kaempferol and the deglycosylated product of sulfated kaempferol-3-glucoside, the sulfation site in sulfated kaempferol-3-glucoside appeared to be the hydroxyl group of the flavonoid skeleton. In addition, by using direct infusion mass spectrometry, it was found that the sulfated product had one sulfonate group within the molecule. These results indicated that AtSULT202B7 functions as a flavonoid glycoside 7-sulfotransferase.

Keywords: Arabidopsis thaliana; AtSULT202B7; flavonoid glycoside; sulfation; sulfotransferase.

Fig. 1

Amino acid sequence comparison of AtSULT202B7, AtSULT202B5 and AtSULT202B1. Identical residues conserved among at least 2 of the 3 enzymes are indicated by black background, and similar residues are indicated in grey. The NAAC (the N-terminal acidic amino acid cluster), which might act as a potencial sorting determinant, is underlined. The 5′-PSB loop (including the conserved lysine residue in the N terminal), which interacts with the 5′-phosphate of PAP, and the 3′-PB motif, for the binding of the 3′-phosphate of PAP, are underlined. The conserved P-loop related motif (GXXGXXK) is also underlined. The catalytic histidine residue, which is highly conserved in almost all known SULTs, is indicated by an arrow.

Fig. 2

SDS gel electrophoretic pattern of recombinant AtSULT202B7 at different stages during purification. Samples were subjected to SDS–PAGE, followed by Coomassie blue staining. Lane 1, molecular weight markers; lane 2, BL21 Escherichia coli homogenate prior to IPTG induction; lane 3, BL21 E. coli homogenate after IPTG induction; lane 4, cytosolic fraction of IPTG-induced BL21 E. coli homogenate; lane 5, AtSULT202B7-GST-fusion protein before thrombin cleavage; lane 6, purified AtSULT202B7 after treatment of thrombin.

Fig. 3

A chemical structure of some representative flavonoids used as substrates for this study.

Fig. 4

pH dependency of the sulfating activity of AtSULT202B7 with flavonols and flavonol glucosides as substrates. The enzymatic assays with 10 µM of substrate compounds were carried out under standard assay conditions using different buffer systems.

Fig. 5

HPLC detection of sulfated kaempferol-3-glucoside. (A) Kaempferol; (B) sulfated kaempferol generated using AtSULT202B7; (C) kaempferol-3-glucoside; (D) sulfated kaempferol-3-glucoside generated using AtSULT202B7; (E) sulfated kaempferol-3-glucoside; (F) deglucosylated products of sulfated kaempferol-3-glucoside generated using β-glucosidase.

Fig. 6

MS2 spectrum of the sulfated kaempferol-3-glucoside. MS2 data were obtained from the 527.05 m/z ion as the precursor for high energy collisional dissociation.