Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes

Hiro omi Tamura, Yoriko Morioka, Hiroshi Homma, Michio Matsui

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The ST-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS), ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5'- phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS. These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.

Original languageEnglish
Pages (from-to)309-314
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume341
Issue number2
DOIs
Publication statusPublished - 1997 May 15

Keywords

  • androsterone
  • chimeric proteins
  • cortisol
  • dehydroepiandrostetone
  • hydroxysteroid
  • rat liver
  • sulfotransferase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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