The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

Abstract

The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3av-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located.That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal glycine of an invariably conserved GXXGXXK motif present in all steroid and phenol sulfotransferases for which primary structures are known. This consistency strongly suggests that all steroid and phenol sulfotransferase genes will be similarly spliced. The GXXGXXK motif forms the active binding site for the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate. Amino acid sequence alignment of 19 cloned steroid and phenol sulfotransferases starting with the GXXGXXK motif indicates that the 3'-terminal exon for each steroid and phenol sulfotransferase gene encodes a similarly sized C-terminal fragment of the protein. Interestingly, on further analysis of the alignment, three distinct amino acid sequence patterns emerge. The presence of the conserved functional GXXGXXK motif suggests that the protein domains encoded by steroid and phenol sulfotransferase 3'-terminal exons have evolved from a common ancestor. Furthermnore, it is hypothesized that during the course of evolution, the 3'-terminal exon further diverged into at least three sulfotransferase subdivisions: a phenol or aryl group, an estrogen or phenolic steroid group, and a neutral steroid group.

Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis.

“…This motif is positioned within a highly conserved region located near the C termini of all steroid and phenol(aryl) sulfotransferases for which primary structures are known (3). Interestingly, the first glycine in the GXXGXXK motif is located at the 5'-splice site for the 3'-terminal exon of gpEST (exon 8), a pattern consistent for all steroid sulfotransferase gene structures cloned to date (4).…”

Molecular Cloning and Expression of cDNA Encoding Human 3′-Phosphoadenylylsulfate:galactosylceramide 3′-Sulfotransferase

“…Hashimoto et al (15) noted a putative PAPS binding motif in sulfotransferases, GXXGXXK, which resembles the P-loop nucleotide binding motif for ATP-and GTP-binding proteins, and this motif is actually conserved near the COOH terminus of many sulfotransferases cloned to date (20). However, there was no such sequence in GalCer sulfotransferase.…”

Steroid Sulfotransferases

“…This approach reveals that in addition to residues gly260, gly263, and Iys266, which are essential for PAPS binding, gly260 and asn267 have a profound effect on the ability of gpEST to efficiently sulfonate the estrogen substrate. On the basis of these findings it is proposed that the sequence GISGDWKN, encoded by the 5'-end of the 3'-terminal exon [exon 8 (92)] represents an essential component of the active site for gpEST and that, inasmuch as the GxxGxxK motif is universally conserved, this area will also prove to be catalytically important for all steroid as well as phenol /aryl sulfotransferases.…”

“…Gene cloning. The complete structural genes for gpEST (92) and hEST (93) have been cloned. The gpEST gene spans 18.6 kb, while the hEST gene is approximately 20 kb in length.…”