Structure-Function Relationships in the Stereospecific and Manganese-dependent 3,4-Dihydroxyphenylalanine/ Tyrosine-sulfating Activity of Human Monoamine-form Phenol Sulfotransferase, SULT1A3

Abstract

The human monoamine-form phenol sulfotransferase (PST), SULT1A3, has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is stereospecific for their D-form enantiomers and can be stimulated dramatically by Mn 2؉ . This activity is not present in the simple phenol-form PST, SULT1A1, which is otherwise >93% identical to SULT1A3 in amino acid sequence. The majority of the differences between these two proteins reside in two variable regions of their sequences. Through the characterization of chimeric PSTs where these two regions were exchanged between them, it was demonstrated that variable Region II of SULT1A3 is required for the stereospecificity of its Dopa/tyrosine-sulfating activity, whereas variable Region I of SULT1A3 is required for the stimulation by Mn 2؉ of this activity. Further studies using point-mutated SULT1A3s mutated at amino acid residues in these two regions and deletional mutants missing residues 84 -86 and 84 -90 implicate residue Glu-146 (in variable Region II of SULT1A3), as well as the presence of residues 84 -90 of variable Region I, in the stereospecificity in the absence of Mn 2؉ . Residue Asp-86 (in variable Region I of SULT1A3), on the other hand, is critical in the Mn 2؉ stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. A model is proposed, with reference to the reported x-ray crystal structure of SULT1A3, to explain how the normal role of SULT1A3 in dopamine regulation may be subverted in the presence of Mn 2؉ . These studies could be relevant in understanding the stereoselective action of SULT1A3 on chiral drugs.The sulfotransferases (STs), 1 which are ubiquitous in both plants and animals, catalyze the sulfation of hydroxyl or amino groups on a variety of target acceptor molecules (1, 2). These enzymes all use adenosine 3Ј-phosphate,5Ј-phosphosulfate (PAPS) as the sulfonyl group donor (3) and share sequences responsible for PAPS binding (4). Although the membranebound STs use proteins, glycolipids, and other macromolecules as acceptor substrates, the cytosolic STs sulfate smaller molecules and are part of the Phase II detoxification pathway for the biotransformation/excretion of drugs and xenobiotics (1, 2). Increasingly, the cytosolic STs have also been shown to be important in regulating the levels and/or activities of endogenous compounds such as thyroid and steroid hormones, catecholamines, and bile acids (5, 6).Based on their sequences, the cytosolic STs have been classified into several gene families (4). Two human cytosolic STs, the monoamine-form and the simple phenol-form phenol sulfotransferases, named SULT1A3 and SULT1A1, respectively (4), show an extensive (Ͼ93%) identity in their amino acid sequences (cf. Fig. 1A) and yet vary widely in their substrate specificity and other properties (7-9). They have thus served as an ideal model system to study structure/function relationships in these proteins. Examination of their aligned sequences revealed that most of the differences between SULT1A3 and SULT1A1 occur in two variable regions, ...

Active Site Mutations and Substrate Inhibition in Human Sulfotransferase 1A1 and 1A3

“…This interaction would prevent binding of the second dopamine molecule by removing a major protein interaction. This is further supported by the fact that Glu-89 is replaced by isoleucine in SULT1A1, this protein shows very low substrate binding affinity for dopamine, and its activity is unaffected by adding Mn 2ϩ (44,45). The mutation of Glu-89 in SULT1A3 to isoleucine results in reduced activity toward dopamine (42,45).…”

“…This is further supported by the fact that Glu-89 is replaced by isoleucine in SULT1A1, this protein shows very low substrate binding affinity for dopamine, and its activity is unaffected by adding Mn 2ϩ (44,45). The mutation of Glu-89 in SULT1A3 to isoleucine results in reduced activity toward dopamine (42,45). In the SULT1A3 crystal structure (15), residues 86 -90 form a mobile loop that intercalates into the active site of a symmetry-related monomer.…”

“…Mn 2ϩ has shown to stimulate dopamine sulfonation by SULT1A3, and this is proposed to occur via interaction between Mn 2ϩ and Asp-86 which is in the mobile loop having residues 86 -90 (44,45). Further, at higher concentration of Mn 2ϩ , it appeared that there was no substrate inhibition observed for dopamine.…”

Regioselective Sulfation and Glucuronidation of Phenolics: Insights into the Structural Basis

“…SULT1A3 has a unique dopa (3,4-dihydroxyphenylalanine; 17 , Fig.1)-sulfating activity that was stereo-selective for their D-form enantiomers [38]. Although there is a lack of experimentally derived differentiation between the formation rates of different sulfated metabolites, molecular docking of D-dopa to the SULT1A3 crystal structure rendered the 3-OH group of D-dopa at a closer position to the catalytic residues than 4-OH, suggesting that 3-O-sulfate should be preferentially generated by SULT1A3 [17].…”

Influence of Phenylalanines 77 and 138 on the Stereospecificity of Aryl Sulfotransferase Iv

“…In this case, the enhanced specificity of SULT1A3 for catecholamines relative to 4-nitrophenol and the reverse specificity with SULT1A1 were linked to a glutamate residue (Glu146) in SULT1A3 that corresponds to an alanine in SULT1A1 (Dajani et al, 1998(Dajani et al, , 1999Brix et al, 1999). Additional investigations on SULT1A1 and SULT1A3 have indicated that Glu146 as well as residues 84 -90 are implicated in the stereospecific sulfation of the D-isomers of DOPA and tyrosine (Pai et al, 2003).…”