title: Structural characterization of human aryl sulphotransferases.
abstract: Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44-107) and B (amino acids 132-164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3'-phosphoadenosine 5'-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87-P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.
authors.affiliation: Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland, 4072, Australia.
authors.forename: L A
authors.initials: LA
authors.lastname: Brix
authors.forename: R G
authors.initials: RG
authors.lastname: Duggleby
authors.forename: A
authors.initials: A
authors.lastname: Gaedigk
authors.forename: M E
authors.initials: ME
authors.lastname: McManus
mesh_terms: D000595:Amino Acid Sequence; D015239:Arylsulfotransferase / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D009596:Nitrophenols; D011993:Recombinant Fusion Proteins; D010724:Phosphoadenosine Phosphosulfate; D015239:Arylsulfotransferase; D004298:Dopamine; C024836:4-nitrophenol