Origins of stereoselectivity in evolved ketoreductases

  1. Noey, E.L. 2
  2. Tibrewal, N. 2
  3. Jiménez-Osés, G. 2
  4. Osuna, S. 2
  5. Park, J. 2
  6. Bond, C.M. 2
  7. Cascio, D. 2
  8. Liang, J. 1
  9. Zhang, X. 1
  10. Huisman, G.W. 1
  11. Tang, Y. 2
  12. Houk, K.N. 22
  1. 1 Codexis Inc., Redwood City, CA, United States
  2. 2 University of California Los Angeles
    info

    University of California Los Angeles

    Los Ángeles, Estados Unidos

    ROR https://ror.org/046rm7j60

Revista:
Proceedings of the National Academy of Sciences of the United States of America

ISSN: 0027-8424

Año de publicación: 2015

Volumen: 112

Número: 51

Páginas: E7065-E7072

Tipo: Artículo

DOI: 10.1073/PNAS.1507910112 SCOPUS: 2-s2.0-84952705870 GOOGLE SCHOLAR

Otras publicaciones en: Proceedings of the National Academy of Sciences of the United States of America

Proyectos relacionados

Financiación Adicional RAMÓN Y CAJAL G.J.O.

2015/00062/001

Resumen

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP+-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.