Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Jan 15;337(Pt 2):337–343.

Structural characterization of human aryl sulphotransferases.

L A Brix 1, R G Duggleby 1, A Gaedigk 1, M E McManus 1
PMCID: PMC1219970  PMID: 9882633

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.

Full Text

The Full Text of this article is available as a PDF (173.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Borchardt R. T., Schasteen C. S. Phenol-sulfotransferase inactivation by 2,3-butanedione and phenylglyoxal: evidence for an active site arginyl residue. Biochem Biophys Res Commun. 1977 Oct 10;78(3):1067–1073. doi: 10.1016/0006-291x(77)90529-0. [DOI] [PubMed] [Google Scholar]
  2. Borchardt R. T., Schasteen C. S., Wu S. E. Phenol sulfotransferase. II. Inactivation by phenylglyoxal, N-ethylmaleimide and ribonucleotide 2',3'-dialdehydes. Biochim Biophys Acta. 1982 Nov 19;708(3):280–293. doi: 10.1016/0167-4838(82)90438-1. [DOI] [PubMed] [Google Scholar]
  3. Brix L. A., Nicoll R., Zhu X., McManus M. E. Structural and functional characterisation of human sulfotransferases. Chem Biol Interact. 1998 Feb 20;109(1-3):123–127. doi: 10.1016/s0009-2797(97)00126-9. [DOI] [PubMed] [Google Scholar]
  4. Coughtrie M. W. Sulphation catalysed by the human cytosolic sulphotransferases--chemical defence or molecular terrorism? Hum Exp Toxicol. 1996 Jul;15(7):547–555. doi: 10.1177/096032719601500701. [DOI] [PubMed] [Google Scholar]
  5. Driscoll W. J., Komatsu K., Strott C. A. Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12328–12332. doi: 10.1073/pnas.92.26.12328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Duffel M. W., Jakoby W. B. On the mechanism of aryl sulfotransferase. J Biol Chem. 1981 Nov 10;256(21):11123–11127. [PubMed] [Google Scholar]
  7. Duggleby R. G. Regression analysis of nonlinear Arrhenius plots: an empirical model and a computer program. Comput Biol Med. 1984;14(4):447–455. doi: 10.1016/0010-4825(84)90045-3. [DOI] [PubMed] [Google Scholar]
  8. Falany C. N. Enzymology of human cytosolic sulfotransferases. FASEB J. 1997 Mar;11(4):206–216. doi: 10.1096/fasebj.11.4.9068609. [DOI] [PubMed] [Google Scholar]
  9. Foldes A., Meek J. L. Rat brain phenolsulfotransferase: partial purification and some properties. Biochim Biophys Acta. 1973 Dec 19;327(2):365–374. doi: 10.1016/0005-2744(73)90419-1. [DOI] [PubMed] [Google Scholar]
  10. Gaedigk A., Lekas P., Berchuk M., Grant D. M. Novel sulfotransferases cloned by RT-PCR: real proteins or PCR artifacts? Chem Biol Interact. 1998 Feb 20;109(1-3):43–52. doi: 10.1016/s0009-2797(97)00119-1. [DOI] [PubMed] [Google Scholar]
  11. Homma H., Ogawa K., Hirono K., Morioka Y., Hirota M., Tanahashi I., Matsui M. Site-directed mutagenesis of rat hepatic hydroxysteroid sulfotransferases. Biochim Biophys Acta. 1996 Sep 5;1296(2):159–166. doi: 10.1016/0167-4838(96)00065-9. [DOI] [PubMed] [Google Scholar]
  12. Kakuta Y., Pedersen L. G., Carter C. W., Negishi M., Pedersen L. C. Crystal structure of estrogen sulphotransferase. Nat Struct Biol. 1997 Nov;4(11):904–908. doi: 10.1038/nsb1197-904. [DOI] [PubMed] [Google Scholar]
  13. Komatsu K., Driscoll W. J., Koh Y. C., Strott C. A. A P-loop related motif (GxxGxxK) highly conserved in sulfotransferases is required for binding the activated sulfate donor. Biochem Biophys Res Commun. 1994 Nov 15;204(3):1178–1185. doi: 10.1006/bbrc.1994.2587. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Marsolais F., Varin L. Identification of amino acid residues critical for catalysis and cosubstrate binding in the flavonol 3-sulfotransferase. J Biol Chem. 1995 Dec 22;270(51):30458–30463. doi: 10.1074/jbc.270.51.30458. [DOI] [PubMed] [Google Scholar]
  16. Marsolais F., Varin L. Mutational analysis of domain II of flavonol 3-sulfotransferase. Eur J Biochem. 1997 Aug 1;247(3):1056–1062. doi: 10.1111/j.1432-1033.1997.01056.x. [DOI] [PubMed] [Google Scholar]
  17. Pennings E. J., Vrielink R., van Kempen G. M. Kinetics and mechanism of the rat brain phenol sulphotransferase reaction. Biochem J. 1978 Jul 1;173(1):299–307. doi: 10.1042/bj1730299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rikke B. A., Roy A. K. Structural relationships among members of the mammalian sulfotransferase gene family. Biochim Biophys Acta. 1996 Jul 17;1307(3):331–338. doi: 10.1016/0167-4781(96)00065-6. [DOI] [PubMed] [Google Scholar]
  19. Sakakibara Y., Takami Y., Nakayama T., Suiko M., Liu M. C. Localization and functional analysis of the substrate specificity/catalytic domains of human M-form and P-form phenol sulfotransferases. J Biol Chem. 1998 Mar 13;273(11):6242–6247. doi: 10.1074/jbc.273.11.6242. [DOI] [PubMed] [Google Scholar]
  20. Tamura H., Morioka Y., Homma H., Matsui M. Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes. Arch Biochem Biophys. 1997 May 15;341(2):309–314. doi: 10.1006/abbi.1997.9979. [DOI] [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Varin L., Ibrahim R. K. Novel flavonol 3-sulfotransferase. Purification, kinetic properties, and partial amino acid sequence. J Biol Chem. 1992 Jan 25;267(3):1858–1863. [PubMed] [Google Scholar]
  23. Varin L., Marsolais F., Brisson N. Chimeric flavonol sulfotransferases define a domain responsible for substrate and position specificities. J Biol Chem. 1995 May 26;270(21):12498–12502. doi: 10.1074/jbc.270.21.12498. [DOI] [PubMed] [Google Scholar]
  24. Varin L., Marsolais F., Richard M., Rouleau M. Sulfation and sulfotransferases 6: Biochemistry and molecular biology of plant sulfotransferases. FASEB J. 1997 Jun;11(7):517–525. doi: 10.1096/fasebj.11.7.9212075. [DOI] [PubMed] [Google Scholar]
  25. Veronese M. E., Burgess W., Zhu X., McManus M. E. Functional characterization of two human sulphotransferase cDNAs that encode monoamine- and phenol-sulphating forms of phenol sulphotransferase: substrate kinetics, thermal-stability and inhibitor-sensitivity studies. Biochem J. 1994 Sep 1;302(Pt 2):497–502. doi: 10.1042/bj3020497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Weinshilboum R. M., Otterness D. M., Aksoy I. A., Wood T. C., Her C., Raftogianis R. B. Sulfation and sulfotransferases 1: Sulfotransferase molecular biology: cDNAs and genes. FASEB J. 1997 Jan;11(1):3–14. [PubMed] [Google Scholar]
  27. Zhang H., Varlamova O., Vargas F. M., Falany C. N., Leyh T. S., Varmalova O. Sulfuryl transfer: the catalytic mechanism of human estrogen sulfotransferase. J Biol Chem. 1998 May 1;273(18):10888–10892. doi: 10.1074/jbc.273.18.10888. [DOI] [PubMed] [Google Scholar]
  28. Zhu X., Veronese M. E., Bernard C. C., Sansom L. N., McManus M. E. Identification of two human brain aryl sulfotransferase cDNAs. Biochem Biophys Res Commun. 1993 Aug 31;195(1):120–127. doi: 10.1006/bbrc.1993.2018. [DOI] [PubMed] [Google Scholar]
  29. Zhu X., Veronese M. E., Iocco P., McManus M. E. cDNA cloning and expression of a new form of human aryl sulfotransferase. Int J Biochem Cell Biol. 1996 May;28(5):565–571. doi: 10.1016/1357-2725(95)00164-6. [DOI] [PubMed] [Google Scholar]
  30. Zhu X., Veronese M. E., Sansom L. N., McManus M. E. Molecular characterisation of a human aryl sulfotransferase cDNA. Biochem Biophys Res Commun. 1993 Apr 30;192(2):671–676. doi: 10.1006/bbrc.1993.1467. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES