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Structural and functional studies of a trans-acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α- and β-ketoreduction

Authors

  • Shawn K. Piasecki,

    1. Institute for Cellular and Molecular Biology, The University of Texas at Austin, Texas
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    • Shawn K. Piasecki and Jianting Zheng contributed equally to this work.

  • Jianting Zheng,

    1. Department of Chemistry and Biochemistry, The University of Texas at Austin, Texas
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    • Shawn K. Piasecki and Jianting Zheng contributed equally to this work.

  • Abram J. Axelrod,

    1. Department of Chemistry and Biochemistry, The University of Texas at Austin, Texas
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  • Madeline E. Detelich,

    1. Department of Chemistry and Biochemistry, The University of Texas at Austin, Texas
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  • Adrian T. Keatinge-Clay

    Corresponding author
    1. Institute for Cellular and Molecular Biology, The University of Texas at Austin, Texas
    2. Department of Chemistry and Biochemistry, The University of Texas at Austin, Texas
    • Correspondence to: Adrian Keatinge-Clay, UT Austin Chemistry, 1 University Station A5300, Austin, TX 78712. E-mail: adriankc@utexas.edu

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  • Accession Numbers: Structure coordinates were deposited in the Protein Data Bank under accession codes 4J1S (apo) and 4J1Q (with NADP+).

  • Conflict of Interest: The authors have no conflict of interest to declare.

  • *Both authors are equally contributed

ABSTRACT

While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This KR naturally reduces both α- and β-keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N-acetylcysteamine-bound β-keto substrate to a D-β-hydroxy product, but also an N-acetylcysteamine-bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a KR that naturally reduces a β-keto group within a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase KRs reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase KRs, is predictive of the orientation of the resulting β-hydroxyl group. Proteins 2014; 82:2067–2077. © 2014 Wiley Periodicals, Inc.

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