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Title: Origins of stereoselectivity in evolved ketoreductases

Abstract

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. Our 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 intermediatemore » for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [2];  [3];  [4];  [4];  [4];  [5];  [5]
+ Show Author Affiliations
  1. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
  2. Univ. of California, Los Angeles, CA (United States). Dept. of Chemical and Biomolecular Engineering
  3. Univ. of California, Los Angeles, CA (United States). Molecular Biology Inst.
  4. Codexis Inc., Redwood City, CA (United States)
  5. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry and Dept. of Chemical and Biomolecular Engineering
Publication Date:
Research Org.:
Univ. of California, Los Angeles, CA (United States); Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE; National Institutes of Health (NIH); National Science Foundation (NSF); Ministry of Economy and Enterprise (MINECO); European Commission (EC)
OSTI Identifier:
1469099
Grant/Contract Number:  
AC02-06CH11357; P41 RR015301; P41 GM103403; OCI-1053575; FC03-02ER63421; GM036700; GM097200; GM075962; JCI-2012-14438; PCIG14-GA-2013-630978; T32 GM067555-11
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 51; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; directed evolution; crystallographic structures; molecular dynamics; theozyme; enantioselectivity

Citation Formats

Noey, Elizabeth L., Tibrewal, Nidhi, Jiménez-Osés, Gonzalo, Osuna, Sílvia, Park, Jiyong, Bond, Carly M., Cascio, Duilio, Liang, Jack, Zhang, Xiyun, Huisman, Gjalt W., Tang, Yi, and Houk, Kendall N. Origins of stereoselectivity in evolved ketoreductases. United States: N. p., 2015. Web. doi:10.1073/pnas.1507910112.
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Noey, Elizabeth L., Tibrewal, Nidhi, Jiménez-Osés, Gonzalo, Osuna, Sílvia, Park, Jiyong, Bond, Carly M., Cascio, Duilio, Liang, Jack, Zhang, Xiyun, Huisman, Gjalt W., Tang, Yi, & Houk, Kendall N. Origins of stereoselectivity in evolved ketoreductases. United States. https://doi.org/10.1073/pnas.1507910112
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Noey, Elizabeth L., Tibrewal, Nidhi, Jiménez-Osés, Gonzalo, Osuna, Sílvia, Park, Jiyong, Bond, Carly M., Cascio, Duilio, Liang, Jack, Zhang, Xiyun, Huisman, Gjalt W., Tang, Yi, and Houk, Kendall N. Mon . "Origins of stereoselectivity in evolved ketoreductases". United States. https://doi.org/10.1073/pnas.1507910112. https://www.osti.gov/servlets/purl/1469099.
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@article{osti_1469099,
title = {Origins of stereoselectivity in evolved ketoreductases},
author = {Noey, Elizabeth L. and Tibrewal, Nidhi and Jiménez-Osés, Gonzalo and Osuna, Sílvia and Park, Jiyong and Bond, Carly M. and Cascio, Duilio and Liang, Jack and Zhang, Xiyun and Huisman, Gjalt W. and Tang, Yi and Houk, Kendall N.},
abstractNote = {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. Our 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.},
doi = {10.1073/pnas.1507910112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 51,
volume = 112,
place = {United States},
year = {Mon Dec 07 00:00:00 EST 2015},
month = {Mon Dec 07 00:00:00 EST 2015}
}
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https://doi.org/10.1073/pnas.1507910112
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Works referenced in this record:

Application of Designed Enzymes in Organic Synthesis
journal, July 2011

  • Strohmeier, Gernot A.; Pichler, Harald; May, Oliver
  • Chemical Reviews, Vol. 111, Issue 7
  • DOI: 10.1021/cr100386u

Asymmetric hydrogenation of cycloalkanones catalyzed by BINAP-iridium(I)-aminophosphine systems
journal, April 1993

  • Zhang, Xiaoyong; Taketomi, Takanao; Yoshizumi, Takashi
  • Journal of the American Chemical Society, Vol. 115, Issue 8
  • DOI: 10.1021/ja00061a036

Advances in the Enzymatic Reduction of Ketones
journal, December 2007

  • Moore, Jeffrey C.; Pollard, David J.; Kosjek, Birgit
  • Accounts of Chemical Research, Vol. 40, Issue 12
  • DOI: 10.1021/ar700167a

Atomic Resolution Structures of R-specific Alcohol Dehydrogenase from Lactobacillus brevis Provide the Structural Bases of its Substrate and Cosubstrate Specificity
journal, June 2005

  • Schlieben, Nils Helge; Niefind, Karsten; Müller, Jörg
  • Journal of Molecular Biology, Vol. 349, Issue 4
  • DOI: 10.1016/j.jmb.2005.04.029

Crystallographic Evidence for Tyr 157 Functioning as the Active Site Base in Human UDP−Galactose 4-Epimerase † , ‡
journal, May 2000

  • Thoden, James B.; Wohlers, Travis M.; Fridovich-Keil, Judith L.
  • Biochemistry, Vol. 39, Issue 19
  • DOI: 10.1021/bi000215l

Cloning, expression, and characterization of an ( R )-specific alcohol dehydrogenase from Lactobacillus kefir
journal, January 2006

Free-Energy Perturbation Simulation on Transition States and Redesign of Butyrylcholinesterase
journal, March 2009

Enantioselective Enzymes by Computational Design and In Silico Screening
journal, February 2015

  • Wijma, Hein J.; Floor, Robert J.; Bjelic, Sinisa
  • Angewandte Chemie International Edition, Vol. 54, Issue 12
  • DOI: 10.1002/anie.201411415

Comparison of simple potential functions for simulating liquid water
journal, July 1983

  • Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.
  • The Journal of Chemical Physics, Vol. 79, Issue 2
  • DOI: 10.1063/1.445869

The Role of Aryne Distortions, Steric Effects, and Charges in Regioselectivities of Aryne Reactions
journal, October 2014

  • Medina, Jose M.; Mackey, Joel L.; Garg, Neil K.
  • Journal of the American Chemical Society, Vol. 136, Issue 44
  • DOI: 10.1021/ja5099935

Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction Cycle
journal, November 2008

  • Smith, Adam J. T.; Müller, Roger; Toscano, Miguel D.
  • Journal of the American Chemical Society, Vol. 130, Issue 46
  • DOI: 10.1021/ja803213p

The role of distant mutations and allosteric regulation on LovD active site dynamics
journal, April 2014

  • Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue
  • Nature Chemical Biology, Vol. 10, Issue 6
  • DOI: 10.1038/nchembio.1503

Engineering the third wave of biocatalysis
journal, May 2012

  • Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.
  • Nature, Vol. 485, Issue 7397
  • DOI: 10.1038/nature11117

The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 Å resolution by X-ray crystallography
journal, June 1999

  • Benach, Jordi; Atrian, Sı́lvia; Gonzàlez-Duarte, Roser
  • Journal of Molecular Biology, Vol. 289, Issue 2
  • DOI: 10.1006/jmbi.1999.2765

Nature of Forces between Large Molecules of Biological Interest*
journal, May 1948

Practical chiral alcohol manufacture using ketoreductases
journal, April 2010

  • Huisman, Gjalt W.; Liang, Jack; Krebber, Anke
  • Current Opinion in Chemical Biology, Vol. 14, Issue 2
  • DOI: 10.1016/j.cbpa.2009.12.003

Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model
journal, April 2003

  • Cossi, Maurizio; Rega, Nadia; Scalmani, Giovanni
  • Journal of Computational Chemistry, Vol. 24, Issue 6
  • DOI: 10.1002/jcc.10189

Enantioselective Enzymes by Computational Design and In Silico Screening
journal, January 2015

  • Wijma, Hein J.; Floor, Robert J.; Bjelic, Sinisa
  • Angewandte Chemie, Vol. 127, Issue 12
  • DOI: 10.1002/ange.201411415

Enantioselective oxazaborolidine reduction of ketones containing heteroatoms
journal, January 1993

Short-chain dehydrogenases/reductases (SDR): the 2002 update
journal, February 2003

Critical Residues for Structure and Catalysis in Short-chain Dehydrogenases/Reductases
journal, April 2002

  • Filling, Charlotta; Berndt, Kurt D.; Benach, Jordi
  • Journal of Biological Chemistry, Vol. 277, Issue 28
  • DOI: 10.1074/jbc.M202160200

Variation on a Theme of SDR
journal, June 2002

The Crystal Structure of R-specific Alcohol Dehydrogenase from Lactobacillus brevis Suggests the Structural Basis of its Metal Dependency
journal, March 2003

Climbing the Density Functional Ladder: Nonempirical Meta–Generalized Gradient Approximation Designed for Molecules and Solids
journal, September 2003

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