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 »
- Authors:
-
- Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
- Univ. of California, Los Angeles, CA (United States). Dept. of Chemical and Biomolecular Engineering
- Univ. of California, Los Angeles, CA (United States). Molecular Biology Inst.
- Codexis Inc., Redwood City, CA (United States)
- 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.
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
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.
@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}
}
Web of Science
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