Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

doi:10.1021/bi6026506

Title: Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

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Abstract

The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographicmore »« less

Authors:: Reger,A.; Carney, J.; Gulick, A.

Publication Date:: Mon Jan 01 00:00:00 EST 2007

Research Org.:: Brookhaven National Laboratory (BNL) National Synchrotron Light Source

Sponsoring Org.:: Doe - Office Of Science

OSTI Identifier:: 930426

Report Number(s):: BNL-81166-2008-JA
Journal ID: ISSN 0006-2960; TRN: US200904%%703

DOE Contract Number:: DE-AC02-98CH10886

Resource Type:: Journal Article

Resource Relation:: Journal Name: Biochemistry; Journal Volume: 46

Country of Publication:: United States

Language:: English

Subject:: 36 MATERIALS SCIENCE; ALANINES; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; ENZYMES; HYPOTHESIS; KINETICS; LEUCINE; LIGASES; LUCIFERASE; MUTANTS; MUTATIONS; PEPTIDES; RESIDUES; ROTATION; SALMONELLA; SPECIFICITY; SUBSTRATES; national synchrotron light source

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                    Reger,A., Carney, J., and Gulick, A. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase.  United States: N. p., 2007. 
        Web.  doi:10.1021/bi6026506.

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                    Reger,A., Carney, J., & Gulick, A. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase.  United States.  doi:10.1021/bi6026506.

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                    Reger,A., Carney, J., and Gulick, A. Mon .  
        "Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase".  United States.  
        doi:10.1021/bi6026506.

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@article{osti_930426,

  title        = {Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase},

  author       = {Reger,A. and Carney, J. and Gulick, A.},

  abstractNote = {The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.},

  doi          = {10.1021/bi6026506},

  journal      = {Biochemistry},

  number       = ,

  volume       = 46,

  place        = {United States},

  year         = {Mon Jan 01 00:00:00 EST 2007},

  month        = {Mon Jan 01 00:00:00 EST 2007}

}

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DOI: 10.1021/bi6026506

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Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea

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We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier worksmore »« less
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Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis

Langan, Patricia S. ; Vandavasi, Venu Gopal ; Cooper, Sarah J. ; ... - ACS Catalysis

Here, the emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of β-lactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and p K _as of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-lactamase in the apo form and an X-ray structure of a Michaelis-like complex withmore »« less
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