title: The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution.
abstract: Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.
authors.forename: Dotan
authors.initials: D
authors.lastname: Amar
authors.forename: Ilana
authors.initials: I
authors.lastname: Berger
authors.forename: Neri
authors.initials: N
authors.lastname: Amara
authors.forename: Gemechu
authors.initials: G
authors.lastname: Tafa
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meijler
authors.forename: Amir
authors.initials: A
authors.lastname: Aharoni
mesh_terms: D001665:Binding Sites; D019020:Directed Molecular Evolution; D004967:Estrogens / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D004967:Estrogens; D050505:Mutant Proteins; D013431:Sulfates; D015238:Sulfotransferases; C020122:estrone sulfotransferase