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Title: Structural Mechanism of Regioselectivity in an Unusual Bacterial Acyl-CoA Dehydrogenase

Abstract

Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an $γ,δ$-ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. While TcsD is homologous to canonical $α,β$-ACADs, it acts regioselectively at the $γ,δ$-position and only on $α,β$-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the $α,β$ position of substrates. From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative $γ,δ$-ACADs that belong to diverse natural product biosynthetic gene clusters. These predictions are supported by the demonstration that a phylogenetically distant homologue of TcsD also regioselectively oxidizesmore » $α,β$-unsaturated substrates. This work exemplifies a powerful approach to understand unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.« less

Authors:
 [1];  [2];  [3];  [4];  [4];  [5];  [5];  [3];  [5];  [5];  [3];  [6];  [6];  [7]; ORCiD logo [6];  [8]; ORCiD logo [9]
  1. Univ. of California, Berkeley, CA (United States). Dept. of Chemistry; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
  5. Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
  6. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Agile BioFoundry, Emeryville, CA (United States). Dept. of Energy
  7. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). QB3 Institute
  8. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division and Molecular Biophysics and Integrated Bioimaging
  9. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). QB3 Institute, Dept. of Chemical & Biomolecular Engineering, and Dept. of Bioengineering; Technical Univ. of Denmark, Horsholm (Denmark). Novo Nordisk Foundation Center for Biosustainability; Shenzhen Inst. for Advanced Technologies (China). Center for Synthetic Biochemistry
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); USDOE Office of Energy Efficiency and Renewable Energy (EERE); National Institutes of Health (NIH); National Science Foundation (NSF)
OSTI Identifier:
1581086
Grant/Contract Number:  
AC02-05CH11231; P30-GM124169-01; DGE-1106400
Resource Type:
Accepted Manuscript
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 142; Journal Issue: 2; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Blake-Hedges, Jacquelyn M., Pereira, Jose Henrique, Cruz-Morales, Pablo, Thompson, Mitchell G., Barajas, Jesus F., Chen, Jeffrey, Krishna, Rohith N., Chan, Leanne Jade G., Nimlos, Danika, Alonso-Martinez, Catalina, Baidoo, Edward E. K., Chen, Yan, Gin, Jennifer W., Katz, Leonard, Petzold, Christopher J., Adams, Paul D., and Keasling, Jay D. Structural Mechanism of Regioselectivity in an Unusual Bacterial Acyl-CoA Dehydrogenase. United States: N. p., 2019. Web. doi:10.1021/jacs.9b09187.
Blake-Hedges, Jacquelyn M., Pereira, Jose Henrique, Cruz-Morales, Pablo, Thompson, Mitchell G., Barajas, Jesus F., Chen, Jeffrey, Krishna, Rohith N., Chan, Leanne Jade G., Nimlos, Danika, Alonso-Martinez, Catalina, Baidoo, Edward E. K., Chen, Yan, Gin, Jennifer W., Katz, Leonard, Petzold, Christopher J., Adams, Paul D., & Keasling, Jay D. Structural Mechanism of Regioselectivity in an Unusual Bacterial Acyl-CoA Dehydrogenase. United States. doi:10.1021/jacs.9b09187.
Blake-Hedges, Jacquelyn M., Pereira, Jose Henrique, Cruz-Morales, Pablo, Thompson, Mitchell G., Barajas, Jesus F., Chen, Jeffrey, Krishna, Rohith N., Chan, Leanne Jade G., Nimlos, Danika, Alonso-Martinez, Catalina, Baidoo, Edward E. K., Chen, Yan, Gin, Jennifer W., Katz, Leonard, Petzold, Christopher J., Adams, Paul D., and Keasling, Jay D. Tue . "Structural Mechanism of Regioselectivity in an Unusual Bacterial Acyl-CoA Dehydrogenase". United States. doi:10.1021/jacs.9b09187.
@article{osti_1581086,
title = {Structural Mechanism of Regioselectivity in an Unusual Bacterial Acyl-CoA Dehydrogenase},
author = {Blake-Hedges, Jacquelyn M. and Pereira, Jose Henrique and Cruz-Morales, Pablo and Thompson, Mitchell G. and Barajas, Jesus F. and Chen, Jeffrey and Krishna, Rohith N. and Chan, Leanne Jade G. and Nimlos, Danika and Alonso-Martinez, Catalina and Baidoo, Edward E. K. and Chen, Yan and Gin, Jennifer W. and Katz, Leonard and Petzold, Christopher J. and Adams, Paul D. and Keasling, Jay D.},
abstractNote = {Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an $γ,δ$-ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. While TcsD is homologous to canonical $α,β$-ACADs, it acts regioselectively at the $γ,δ$-position and only on $α,β$-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the $α,β$ position of substrates. From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative $γ,δ$-ACADs that belong to diverse natural product biosynthetic gene clusters. These predictions are supported by the demonstration that a phylogenetically distant homologue of TcsD also regioselectively oxidizes $α,β$-unsaturated substrates. This work exemplifies a powerful approach to understand unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.},
doi = {10.1021/jacs.9b09187},
journal = {Journal of the American Chemical Society},
number = 2,
volume = 142,
place = {United States},
year = {2019},
month = {12}
}

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This content will become publicly available on December 3, 2020
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