Journal of Molecular Biology
Regular articleCrystal structure of human catecholamine sulfotransferase1
Introduction
The function of biological molecules can be regulated by the transfer and removal of phosphate groups at specific points in the structure, through the action of kinases and phosphatases. Similarly, the addition and removal of sulfate can modify the function of biological molecules through the action of sulfotransferase and sulfatase enzymes. Although the three-dimensional structures of several hundred kinase and phosphatase enzymes, enzyme complexes and enzyme mutants are known, the structures of only two sulfotransferase Kakuta et al 1997, Kakuta et al 1999 and two sulfatase enzymes have been reported Bond et al 1997, Lukatela et al 1998. We have therefore initiated structural studies on sulfotransferases to gain further insight into the structural and functional relationships of these enzymes.
Sulfotransferases are a superfamily of enzymes found in species ranging from bacteria to humans, which catalyse the sulfonation reaction on a variety of endogenous and exogenous substrates. While the substrate specificity of individual sulfotransferases differs significantly, all utilize the same sulfate donor cofactor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The sulfotransferase superfamily is grouped into two major classes that share less than 20 % sequence identity and which differ in terms of their solubility, size, subcellular distribution and substrate specificity. The membrane bound sulfotransferases (45–100 kDa and located in the Golgi apparatus) catalyze the sulfonation of large endogenous molecules such as heparan, glycoproteins, glycosaminoglycans and tyrosyl protein residues Hashimoto et al 1992, Ong et al 1998, Orellana et al 1994, Honke et al 1997, Fukuta et al 1998, Niehrs and Huttner 1990. The cytosolic sulfotransferases (30–35 kDa in size and located in the cell cytoplasm) catalyze the sulfonation of xenobiotics, dietary carcinogens, neurotransmitters and hormones (Falany, 1997). Until recently, sulfonation of these small molecules was regarded as a detoxification pathway leading to the formation of water soluble metabolites that are readily excreted. However, for certain compounds including carcinogens, bioactive peptides and the antihypertensive and hair growth stimulant minoxidil, sulfonation is an activation pathway.
The mammalian cytosolic sulfotransferases are classified based on sequence identity †. The SULT1, SULT2 and SULT3 families share less than 45% sequence identity (Weinshilboum et al., 1997). The SULT1 family is further subdivided into SULT1A, SULT1B, SULT1C and SULT1E. Members within each subfamily share more than 60% sequence identity. Although the sequence identity of the sulfotransferases varies significantly, sequence alignments reveal regions of highly conserved residues (Kakuta et al., 1998a) that have been targeted for mutational studies Kakuta et al 1998b, Komatsu et al 1994, Marsolais and Varin 1995. The recent publication of the first two sulfotransferase crystal structures of the cytosolic mouse estrogen sulfotransferase (SULT1E1) and the sulfotransferase domain of the human membrane-bound N-deacetylase/N-sulfotransferase1 (HSNST-1) that sulfonates heparan shows that these conserved regions interact with the cofactor PAPS Kakuta et al 1997, Kakuta et al 1999.
We now report the crystal structure of a third sulfonating enzyme, the catecholamine sulfotransferase SULT1A3, at 2.4 Å resolution. SULT1A3 (previously called HAST3) is the enzyme primarily responsible for sulfonation of catecholamines such as dopamine, adrenaline and noradrenaline and was originally isolated in our laboratory from human brain Zhu et al 1993, Veronese et al 1994. SULT1A3 does not sulfonate heparan or estrone/estradiol, the preferred substrates of HSNST-1 and SULT1E1, respectively.
Section snippets
Crystallization and structure determination
Crystals of human SULT1A3 grow readily from polyethylene glycol 8000 and lithium sulfate. The enzyme used for crystallization was pre-incubated with the reaction product inhibitor PAP (adenosine 3′, 5′-diphosphate) prior to crystallization, but density at the active site indicates that sulfate is bound rather than PAP (see below). SULT1A3 crystals diffract to a limiting resolution of 2.4 Å and belong to the hexagonal space group P3221 with unit cell dimensions a, b=56.4 Å and c=191.1 Å and one
Discussion
Sulfotransferases are a superfamily of enzymes that catalyze the sulfonation of a wide variety of structurally and chemically diverse compounds. Until now, structural information has been available for only two of these enzymes, mouse SULT1E1 and human HSNST-1 which differ significantly from SULT1A3 in both their primary sequence and substrate specificities. Our structural comparison of SULT1A3 with SULT1E1 and HSNST-1 reveals that the enzymes incorporate a common structural fold consistent
Expression, purification and crystallization
Recombinant SULT1A3 was expressed as described by Gaedigk et al. (1998) and bacterial cytosol was prepared by the method described by Gillam et al. (1993). SULT1A3 protein was purified using DEAE Sepharose CL-6B chromatography as described by Falany et al. (1989). Partially purified protein was dialyzed against buffer A (5 mM NaPO4 (pH 6.8), 0.25 M sucrose, 10 % (v/v) glycerol) and applied to a Macro-Prep Ceramic Hydroxyapatite type I column (BioRad Laboratories, Hercules, CA, USA) which had
Acknowledgements
We thank Alun Jones for help with mass spectrometric data measurement and analysis, Dr Luke Guddat for help with X-ray data measurement and Joel Tyndall for help with Figures. We also acknowledge Dr Elizabeth Gillam for advice on the purification of SULT1A3. The SULT1A3 research is supported by grants from the National Health and Medical Research Council of Australia and the University of Queensland (Mayne Bequest Fund). This work is based on research conducted at the Stanford Synchrotron
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2019, Biochemical PharmacologyCitation Excerpt :Interestingly, none of the cynomolgus SULTs showed substantial catalytic selectivity with the substrates used under the present conditions (in the presence of 1 mM 3′-phosphoadenosine-5′-phosphosulfate), unlike human SULTs, although cynomolgus SULTs did show variable activities towards each substrate (Fig. 7B). In this study, because of reported important residues 106–108 and 225–259 among 295 amino acids for substrate and cofactor binding sites, respectively, we assumed that the 6 × histidine-tags at the N-terminus of recombinant cynomolgus SULTs would have little or minor effects on catalytic activities of crystalized human SULT1A1 [23] and SULT1A3 [24]. Conjugation activities of cynomolgus SULTs in livers were highly variable (Fig. 8B).
Effects of human SULT1A3/SULT1A4 genetic polymorphisms on the sulfation of acetaminophen and opioid drugs by the cytosolic sulfotransferase SULT1A3
2018, Archives of Biochemistry and BiophysicsCitation Excerpt :Several crystal structures of the human SULT1A3 have been reported [33,45,46]. These studies have unveiled structural elements that are important in the catalysis (residue His108), the PAPS-binding (residues 45TYPKSGTT52, Arg130, Ser138, and 257RKG259), the substrate-binding/specificity (residues Asp86 and Glu146) [33], the N-terminal βA- and βB-sheets (residues Leu12-Val15 and Val18-Ile21, respectively) important in the polypeptide folding [46,47], and the C-terminal dimerization motif (residues Lys265-Glu274, with a sequence motif KXXXTVXXXE) [48]. Six of the twelve SULT1A3 allozymes analyzed contain amino acid variations in the N-terminal region encompassing the above-mentioned βA- and βB-sheets, which have been proposed to be important in the polypeptide chain folding [47].
Sulfation of catecholamines and serotonin by SULT1A3 allozymes
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