Sulfation of catecholamines and serotonin by SULT1A3 allozymes

Abstract

Previous studies have demonstrated the involvement of sulfoconjugation in the metabolism of catecholamines and serotonin. The current study aimed to clarify the effects of single nucleotide polymorphisms (SNPs) of human SULT1A3 and SULT1A4 genes on the enzymatic characteristics of the sulfation of dopamine, epinephrine, norepinephrine and serotonin by SULT1A3 allozymes. Following a comprehensive search of different SULT1A3 and SULT1A4 genotypes, twelve non-synonymous (missense) coding SNPs (cSNPs) of SULT1A3/SULT1A4 were identified. cDNAs encoding the corresponding SULT1A3 allozymes, packaged in pGEX-2T vector were generated by site-directed mutagenesis. SULT1A3 allozymes were expressed, and purified. Purified SULT1A3 allozymes exhibited differential sulfating activity toward catecholamines and serotonin. Kinetic analyses demonstrated differences in both substrate affinity and catalytic efficiency of the SULT1A3 allozymes. Collectively, these findings provide useful information relevant to the differential metabolism of dopamine, epinephrine, norepinephrine and serotonin through sulfoconjugation in individuals having different SULT1A3/SULT1A4 genotypes.

Keywords: Catecholamines; Cytosolic sulfotransferase; SULT1A3; Serotonin; Single nucleotide polymorphisms; Sulfation.

Figure 1

Amino acid sequence of the human SULT1A3 showing the locations of amino acid residues involved in the SULT1A3/SULT1A4 cSNPs and sequences/residues reported to be involved in PAPS-binding, substrate-binding, and/or catalysis. Residues circled with white background are involved in PAPS-binding. Residues enclosed in square are involved in substrate-binding. Residue enclosed in diamond is involved in catalysis. Residues circled with black background refer to the locations of amino acid substitutions in the polypeptide chain of the SULT1A3 molecule. Residues circled with gray background refer to the substituting amino acids. The figure was generated using Protter, a web tool for interactive protein feature visualization [63].

Figure 2

Ribbon diagram of the structure of human SULT1A3-dopamine-PAP complex showing the locations of amino acid residues involved in the SULT1A3/SULT1A4 cSNPs. The structure of SULT1A3 (Protein Data Bank code: 2A3R [37]) was edited using USCF Chimera, a molecular modeling software [64]. DA and PAP molecules in the structure are shown by bond structures. Loops 1, 2, and 3 refer to Asp66-Met77, Ser228-Gly259, and Lys85-Pro90 segments previously reported to form a gate for substrate entry. Side chains of the amino acid residues involved in the SULT1A3/SULT1A4 cSNPs, Arg9, Pro10, Val15, Val18, Pro101, Arg144, Asn235, Ser290, are indicated by bond structures.

Figure 3

SDS gel electrophoretic pattern of the purified human SULT1A3 allozymes. SDS-PAGE was performed on a 12% gel, followed by Coomassie Blue staining. Samples analyzed in lanes 1 through 13 correspond to SULT1A3-WT (wild-type), SULT1A3-T7P, SULT1A3-S8P, SULT1A3-R9C, SULT1A3-P10L, SULT1A3-V15M, SULT1A3-V18F, SULT1A3-P101L, SULT1A3-P101H, SULT1A3-R144C, SULT1A3-K234N, SULT1A3-N235T and SULT1A3-S290T. Positions of protein molecular weight markers are indicated on the right.

Figure 4

Specific activities of the sulfation of dopamine (DA) by human SULT1A3 allozymes. Concentrations of dopamine used in the enzymatic assays were 0.5 μM (A) and 5.0 μM (B). Specific activity refers to nmol dopamine sulfated/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. WT refers to wild-type SULT1A3.

Figure 5

Specific activities of the sulfation of epinephrine (EP) by human SULT1A3 allozymes. Concentrations of epinephrine used in the enzymatic assays were 1 μM (A) and 10 μM (B). Specific activity refers to nmol epinephrine sulfated/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. WT refers to wild-type SULT1A3.

Figure 6

Specific activities of the sulfation of norepinephrine (NE) by human SULT1A3 allozymes. Concentrations of norepinephrine used in the enzymatic assays were 1 μM (A) and 10 μM (B). Specific activity refers to nmol norepinephrine sulfated/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. WT refers to wild-type SULT1A3.

Figure 7

Specific activities of the sulfation of serotonin (5-HT) by human SULT1A3 allozymes. Concentrations of serotonin used in the enzymatic assays were 10 μM (A) and 100 μM (B). Specific activity refers to nmol serotonin sulfated/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. WT refers to wild-type SULT1A3.

Figure 8

Kinetic analysis for the sulfation of catecholamines and serotonin by wild-type human SULT1A3. Panels (A), (B), (C) and (D) illustrate the Michaelis–Menten saturation curves for the sulfation of dopamine (DA), epinephrine (EP), norepinephrine (NE), and serotonin (5-HT), respectively. The insets show the Lineweaver-Burk plots generated based on the data shown in each of the four panels. Arrow signs indicate the concentrations at which substrate inhibition started taking place. Data shown represent calculated mean ± standard deviation derived from three experiments.