A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

  1. Koch, A.A. 11
  2. Hansen, D.A. 11
  3. Shende, V.V. 11
  4. Furan, L.R. 1
  5. Houk, K.N. 3
  6. Jiménez-Osés, G. 2
  7. Sherman, D.H. 1111
  1. 1 University of Michigan–Ann Arbor
    info

    University of Michigan–Ann Arbor

    Ann Arbor, Estados Unidos

    ROR https://ror.org/00jmfr291

  2. 2 Universidad de La Rioja
    info

    Universidad de La Rioja

    Logroño, España

    ROR https://ror.org/0553yr311

  3. 3 University of California Los Angeles
    info

    University of California Los Angeles

    Los Ángeles, Estados Unidos

    ROR https://ror.org/046rm7j60

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Revista:
Journal of the American Chemical Society

ISSN: 0002-7863

Año de publicación: 2017

Volumen: 139

Número: 38

Páginas: 13456-13465

Tipo: Artículo

DOI: 10.1021/JACS.7B06436 SCOPUS: 2-s2.0-85030111605 WoS: WOS:000412043000030 GOOGLE SCHOLAR

Otras publicaciones en: Journal of the American Chemical Society

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Resumen

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones. © 2017 American Chemical Society.