A new type of sulfation reaction: C-sulfonation for α,β-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1

Abstract Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,β-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to the α-carbon of α,β-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,β-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,β-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.


Contents
Page S-15: Table S2 Page S-16: Table S3 Page S-17: Table S4 Figure Nucleotides are numbered in the 5' to 3' direction with the adenosine of the translation initiation codon designated as +1.The translation stop codon is indicated by an asterisk.SULT7A1 cDNA consists of a 5'-terminal untranslated sequence (5'-UTR) of 115 bp, an open reading frame (ORF) of 870 nucleotides encoding a 290 amino acid polypeptide, a 3'-UTR of 1745 bp with two typical polyadenylation signal sequence (AATAAA), and a poly(A) tail.Two amino acid residues, Ser3 and Gly263, are different from other reference sequences (GenBank accession numbers; BC151094, BC151096, and BC172155).
-S-1-    Samples were subjected to SDS-PAGE on a 12% gel, followed by Coomassie Blue staining.The purification procedure was described in Experimental procedures.Lane 1, before IPTG induction; lane 2, after IPTG induction; lane 3, cytosol portion; lane 4, GSTfusion protein; lane 5, purified enzyme.Table S1 a COSY refers to the correlations observed in correlation spectroscopy (COSY) analysis and the coupling constants were not determined due to the multiple or overlapping proton signals.-S-14-Table S1.NMR data of 15d-PGJ 2 and the sulfonated product.

Figure S1 .
Figure S1.Nucleotide and deduced amino acid sequences of mouse SULT7A1.Nucleotides are numbered in the 5' to 3' direction with the adenosine of the translation initiation codon designated as +1.The translation stop codon is indicated by an asterisk.SULT7A1 cDNA consists of a 5'-terminal untranslated sequence (5'-UTR) of 115 bp, an open reading frame (ORF) of 870 nucleotides encoding a 290 amino acid polypeptide, a 3'-UTR of 1745 bp with two typical polyadenylation signal sequence (AATAAA), and a poly(A) tail.Two amino acid residues, Ser3 and Gly263, are different from other reference sequences (GenBank accession numbers; BC151094, BC151096, and BC172155).

Figure S2 .Figure
Figure S2.Amino acid sequence alignment of human and mouse SULTs.Identical amino acid residues are marked by grey background.Solid lines indicate the 'signature sequences' involved in the binding of PAPS.Arrows indicate the catalytic residue His conserved among all known SULTs.Asterisks indicate unique residues, His51 and Cys234, in the active site of SULT7A1 as shown in Fig. 4b.

Figure S4 .
Figure S4.Kinetics analyses of the sulfonation catalyzed by SULT7A1.Kinetic assay was carried out to perform the enzymatic assays with the different concentrations of 2cyclopentenone (a), 2-cyclohexanone (b), PGA 2 (c), delta12-PGJ 2 (d), 15d-PGJ 2 (e), or PAPS (f) under standard assay conditions as described in Materials and Methods.The fitting curves were generated using Michaelis-Menten kinetics.Eadie-Hofstee plots are inserted under each fitting curve.The data are calculated mean ± SD (n = 3).

Figure S5 .
Figure S5.Detailed MS analysis of the sulfonated product of 2cyclopentenone.a, Isotopic MS spectrum of the sulfonated product of 2cyclopentenone.Intensity of each isotopic spectrum, 13 C (5.55%) or 34 S (4.53%), was expressed in relative values (%) against monoisotopic spectrum at m/z of 163.0061.b, MS/MS spectrum of 34 S-isotopic ion (165.0019m/z).

Figure S7 .Figure S8 .Figure S10 .
Figure S7.Correlation spectroscopy (COSY) analysis of the sulfonated product of 15d-PGJ 2 .Each of protons numbered in chemical structures was assigned for the corresponding spectrum.Correlations among H8, H9, and H10 are represented by the line circles.

Figure S11 .
Figure S11.Analysis of [ 35 S]sulfonated products of prostaglandins.The figure shows the autoradiograph taken from the TLC plate.Lane 1 and 2 correspond to the reaction mixture of in vitro enzyme assay (lane 1) or labeling media of SULT7A1-expressing BHK-21 cells (lane 2).CT refers to the control sample without added prostaglandins.

Figure S12 .
Figure S12.The proposed reaction mechanism of the unconventional sulfonation reaction by SULT7A1.Arrows in the catalytic residue-PAPS-substrate structure indicate the transition of electron pair.

Figure S13 .
Figure S13.Sulfonation of a,b-unsaturated carbonyl compounds by human SULTs and sulfatase assay for [ 35 S]sulfonated products of a,b-unsaturated carbonyl compounds.(a) Specific sulfonating activity of human SULTs toward 2-cyclohexenone.The activity refers to pmol substrate sulfonated/min/mg purified enzyme.ND refers to activity not detected with the detection limit estimated to be 1.0 pmol/min/mg protein.Data represent mean ± SD derived from three determinations.(b) The figure shows the autoradiograph taken from the TLC plate.Lane 1 and 2 correspond to the reaction mixture without sulfatase (lane 1) or with (lane 2).2-CH and 2-CP refers to the 2-cyclohexenone and 2-cyclopentenone used as the substrate, respectively.Arrows indicate the sulfonated derivatives.