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.
Park
B C
BC
Section on Steroid Regulation, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Lee
Y C
YC
Strott
C A
CA
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