Identification by chimera formation and site-selected mutagenesis of a key amino acid residue involved in determining stereospecificity of guinea pig 3-hydroxysteroid sulfotransferase isoforms

Abstract

The 3-hydroxysteroid sulfotransferases that have been isolated and cloned from humans and rodents appear to have broad substrate specificities. In the guinea pig, however, two 3-hydroxysteroid sulfotransferases have been isolated that function according to an innate stereospecificity: the α- isoform acts on steroids with a 3-hydroxyl group oriented in the α position, whereas the β-isoform acts on steroids where the 3-hydroxyl group is in a β orientation. To examine the structural basis for this remarkable stereoselectivity, chimeras of the two enzymes, which are 87% identical, were constructed. A chimera consisting of the NH2-terminal 91 amino acids of the α-isoform and the COOH-terminal 196 amino acids of the β-isoform displayed activity similar to that of the α-isoform. Site-selected mutagenesis of this 3α/β-hydroxysteroid sulfotransferase chimera involving the 12 amino acid differences that exist between the two isoforms within the 91 amino acid NH2-terminal region revealed that the amino acid residue at position 51 plays a fundamental role in determining the stereospecificity exhibited by the α- and β-isoforms, i.e. if residue 51 is an asparagine, α activity predominates, whereas if an isoleucine is in that position, β activity prevails.