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Protein Engineering: -A Practical Example- Convert a sulfotransferase to a dehydratase

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Protein Engineering: -A Practical Example- Convert a sulfotransferase to a dehydratase

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    1. Protein Engineering: -A Practical Example- Convert a sulfotransferase to a dehydratase Student: Wei-Ti Chen Adviser: Yuh-Shyung Yang

    2. The structure of Estrogen Sulfotransferase The main core of the molecule is composed of an a/b motif. The motif contains: *b sheet comprised of five parallel b-strands surrounded by a-helices on both sides. *conserved helix running across the top of the fold.

    3. PAPS-binding Site for hEST

    4. The Mechanism

    5. Retinol Dehydratase (RD) RD catalyzes the transformation of retinol to anhydroretinol, and involved in many biological processes including morphogenesis. RD is most similar to EST, and shares 26% sequence identity.@ RD(41kDa) is larger than EST(35kDa) in due to 25-amino acid N-terminal extension and a 32-amino acid insertion.

    6. Sequence Alignment of RD and hEST

    7. The structure of RD RD is composed of two domains: *Larger domain has a core five-strand parallel b-sheet and eight a-helices. *Smaller domain comprised of a three-stranded antiparallel b-sheet with six a-helices. The lid in RD may move to provide access to the substrate binding cavity, but it also provides amino acids that are important for substrate recognition.

    8. Crystal structure of RD

    9. Retinol Binding Site

    10. Mechanism

    11. Conclusion Retinol binds to RD with its ring nearest the helical lid. Adding the additional lid segment to ST, we can convert ST to hydratase.

    12. My Experience after Reading Before protein engineering: Check the the sequence with others, e.g. superfamily . Check catalytic mechanism with others. After protein engineering: Site-mutagenesis

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