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Title: Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

Abstract

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closuremore » of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.« less

Authors:
 [1];  [1];  [1]
  1. Purdue Univ., West Lafayette, IN (United States). Dept. of Biochemistry
Publication Date:
Research Org.:
Purdue Univ., West Lafayette, IN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1345618
Grant/Contract Number:  
AC02-06CH11357; 085P1000817
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Chemistry
Additional Journal Information:
Journal Volume: 4; Journal ID: ISSN 2296-2646
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; substrate analog; enzyme mechanism; oxyanion hole; substrate-assisted catalysis

Citation Formats

Murphy, Jesse R., Mullins, Elwood A., and Kappock, T. Joseph. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase. United States: N. p., 2016. Web. doi:10.3389/fchem.2016.00023.
Murphy, Jesse R., Mullins, Elwood A., & Kappock, T. Joseph. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase. United States. doi:10.3389/fchem.2016.00023.
Murphy, Jesse R., Mullins, Elwood A., and Kappock, T. Joseph. Mon . "Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase". United States. doi:10.3389/fchem.2016.00023. https://www.osti.gov/servlets/purl/1345618.
@article{osti_1345618,
title = {Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase},
author = {Murphy, Jesse R. and Mullins, Elwood A. and Kappock, T. Joseph},
abstractNote = {Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.},
doi = {10.3389/fchem.2016.00023},
journal = {Frontiers in Chemistry},
number = ,
volume = 4,
place = {United States},
year = {2016},
month = {5}
}

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Works referenced in this record:

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X-Ray Diffraction data from succinyl-CoA:acetate CoA-transferase (EC , source of 5DW4 structure
dataset, May 2016


    Works referencing / citing this record:

    X-Ray Diffraction data from succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18), N347A mutant bound to CoA, source of 5DDK structure
    dataset, May 2016

    • Kappock, T. Joseph; Murphy, Jesse R.; Mullins, Elwood A.
    • SBGrid Data Bank
    • DOI: 10.15785/sbgrid/282

    X-Ray Diffraction data from succinyl-CoA:acetate CoA-transferase (EC , source of 5DW4 structure
    dataset, May 2016


    X-Ray Diffraction data from succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18), source of 5DW5 structure
    dataset, July 2015


    X-Ray Diffraction data from succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18), source of 5E5H structure
    dataset, May 2016


    Long-term continuous evolution of acetate resistantAcetobacter aceti
    journal, August 2003

    • Steiner, Peter; Sauer, Uwe
    • Biotechnology and Bioengineering, Vol. 84, Issue 1
    • DOI: 10.1002/bit.10741

    Understanding the Acid-Base Properties of Adenosine: The Intrinsic Basicities of N1, N3 and N7
    journal, May 2011

    • Kapinos , Larisa E.; Operschall, Bert P.; Larsen, Erik
    • Chemistry - A European Journal, Vol. 17, Issue 29
    • DOI: 10.1002/chem.201003544

    Chemical approaches to detect and analyze protein sulfenic acids: DETECTION AND ANALYSIS OF SULFENIC ACIDS
    journal, September 2013

    • Furdui, Cristina M.; Poole, Leslie B.
    • Mass Spectrometry Reviews, Vol. 33, Issue 2
    • DOI: 10.1002/mas.21384

    On the importance of orientation in general base catalysis by carboxylate
    journal, June 1981


    Protein production by auto-induction in high-density shaking cultures
    journal, May 2005


    A role for flexible loops in enzyme catalysis
    journal, December 2010

    • Malabanan, M. Merced; Amyes, Tina L.; Richard, John P.
    • Current Opinion in Structural Biology, Vol. 20, Issue 6
    • DOI: 10.1016/j.sbi.2010.09.005

    A New Generation of Crystallographic Validation Tools for the Protein Data Bank
    journal, October 2011


    Role of Binding Energy with Coenzyme A in Catalysis by 3-Oxoacid Coenzyme A Transferase
    journal, September 1995

    • Whitty, Adrian; Fierke, Carol A.; Jencks, William P.
    • Biochemistry, Vol. 34, Issue 37
    • DOI: 10.1021/bi00037a005

    Structure of a NADH-Insensitive Hexameric Citrate Synthase that Resists Acid Inactivation
    journal, November 2006

    • Francois, Julie A.; Starks, Courtney M.; Sivanuntakorn, Sasitorn
    • Biochemistry, Vol. 45, Issue 45
    • DOI: 10.1021/bi061083k

    Specificity in Transition State Binding: The Pauling Model Revisited
    journal, February 2013

    • Amyes, Tina L.; Richard, John P.
    • Biochemistry, Vol. 52, Issue 12
    • DOI: 10.1021/bi301491r

    Generation and stability of a simple thiol ester enolate in aqueous solution
    journal, December 1992

    • Amyes, Tina L.; Richard, John P.
    • Journal of the American Chemical Society, Vol. 114, Issue 26
    • DOI: 10.1021/ja00052a028

    Theoretical assessments of the basicity and nucleophilicity of carboxylate syn and anti lone pairs
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    • Li, Yi; Houk, K. N.
    • Journal of the American Chemical Society, Vol. 111, Issue 12
    • DOI: 10.1021/ja00194a059

    Geometrical reaction coordinates. II. Nucleophilic addition to a carbonyl group
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    • Burgi, H. B.; Dunitz, J. D.; Shefter, Eli.
    • Journal of the American Chemical Society, Vol. 95, Issue 15
    • DOI: 10.1021/ja00796a058

    On Catalytic Preorganization in Oxyanion Holes: Highlighting the Problems with the Gas-Phase Modeling of Oxyanion Holes and Illustrating the Need for Complete Enzyme Models
    journal, October 2010

    • Kamerlin, Shina C. L.; Chu, Zhen T.; Warshel, A.
    • The Journal of Organic Chemistry, Vol. 75, Issue 19
    • DOI: 10.1021/jo100651s

    Addgene provides an open forum for plasmid sharing
    journal, April 2012

    • Herscovitch, Melanie; Perkins, Eric; Baltus, Andy
    • Nature Biotechnology, Vol. 30, Issue 4
    • DOI: 10.1038/nbt.2177

    Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli
    journal, June 2009

    • Bennett, Bryson D.; Kimball, Elizabeth H.; Gao, Melissa
    • Nature Chemical Biology, Vol. 5, Issue 8
    • DOI: 10.1038/nchembio.186

    Announcing the worldwide Protein Data Bank
    journal, December 2003

    • Berman, Helen; Henrick, Kim; Nakamura, Haruki
    • Nature Structural & Molecular Biology, Vol. 10, Issue 12
    • DOI: 10.1038/nsb1203-980

    Cloning and Characterization of a Eukaryotic Pantothenate Kinase Gene ( panK ) from Aspergillus nidulans
    journal, January 1999

    • Calder, Robert B.; Williams, Robin S. B.; Ramaswamy, Gayathri
    • Journal of Biological Chemistry, Vol. 274, Issue 4
    • DOI: 10.1074/jbc.274.4.2014

    MOLE 2.0: advanced approach for analysis of biomacromolecular channels
    journal, August 2013

    • Sehnal, David; Svobodová Vařeková, Radka; Berka, Karel
    • Journal of Cheminformatics, Vol. 5, Issue 1
    • DOI: 10.1186/1758-2946-5-39