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.
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 alpha-isoform acts on steroids with a 3-hydroxyl group oriented in the alpha position, whereas the beta-isoform acts on steroids where the 3-hydroxyl group is in a beta 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 NH(2)-terminal 91 amino acids of the alpha-isoform and the COOH-terminal 196 amino acids of the beta-isoform displayed activity similar to that of the alpha-isoform. Site-selected mutagenesis of this 3alpha/beta-hydroxysteroid sulfotransferase chimera involving the 12 amino acid differences that exist between the two isoforms within the 91 amino acid NH(2)-terminal region revealed that the amino acid residue at position 51 plays a fundamental role in determining the stereospecificity exhibited by the alpha- and beta-isoforms, i.e. if residue 51 is an asparagine, alpha activity predominates, whereas if an isoleucine is in that position, beta activity prevails.
Section on Steroid Regulation, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
B C
BC
Park
Y C
YC
Lee
C A
CA
Strott
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000738:Androsterone / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D006168:Guinea Pigs; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011284:Pregnenolone / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011284:Pregnenolone; D000738:Androsterone; D015238:Sulfotransferases; C028152:alcohol sulfotransferase