Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity.
The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure-activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146-->Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in K(m) value) but 54-fold less efficient in sulphating dopamine (8-fold increase in K(m) value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.
Tsoi
Carrie
C
Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden. Carr_ts@yahoo.se
Widersten
Mikael
M
Morgenstern
Ralf
R
Swedmark
Stellan
S
D000596:Amino Acids; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D004285:Dogs; D004298:Dopamine / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009596:Nitrophenols / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D009596:Nitrophenols; D011994:Recombinant Proteins; D001224:Aspartic Acid; C431463:SULT1D1 sulfotransferase; D015238:Sulfotransferases; D004298:Dopamine; C024836:4-nitrophenol