A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure

Filippova, Ekaterina V.; Kuhn, Misty L.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Ballicora, Miguel A.; Anderson, Wayne F.

doi:10.1016/j.jmb.2015.01.009

Title: A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure

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Abstract

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families ofmore »« less

Authors:: Filippova, Ekaterina V.; Kuhn, Misty L.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Ballicora, Miguel A.; Anderson, Wayne F.

Publication Date:: Sun Mar 01 00:00:00 EST 2015

Research Org.:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Org.:: USDOE Office of Science (SC); National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID); National Science Foundation (NSF)

OSTI Identifier:: 1233990

Alternate Identifier(s):: OSTI ID: 1196374; OSTI ID: 1225226; OSTI ID: 1391850

Grant/Contract Number:: AC02-06CH11357; HHSN272200700058C; HHSN272201200026C; MCB 1024945

Resource Type:: Published Article

Journal Name:: Journal of Molecular Biology

Additional Journal Information:: Journal Name: Journal of Molecular Biology Journal Volume: 427 Journal Issue: 6 PB; Journal ID: ISSN 0022-2836

Publisher:: Elsevier

Country of Publication:: United Kingdom

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; spermidine/spermine; dodecamer; allosteric enzyme; GNAT; acetyltransferase

Citation Formats


                    Filippova, Ekaterina V., Kuhn, Misty L., Osipiuk, Jerzy, Kiryukhina, Olga, Joachimiak, Andrzej, Ballicora, Miguel A., and Anderson, Wayne F. A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure.  United Kingdom: N. p., 2015. 
Web.  doi:10.1016/j.jmb.2015.01.009.

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                    Filippova, Ekaterina V., Kuhn, Misty L., Osipiuk, Jerzy, Kiryukhina, Olga, Joachimiak, Andrzej, Ballicora, Miguel A., & Anderson, Wayne F. A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure.  United Kingdom.  https://doi.org/10.1016/j.jmb.2015.01.009

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                    Filippova, Ekaterina V., Kuhn, Misty L., Osipiuk, Jerzy, Kiryukhina, Olga, Joachimiak, Andrzej, Ballicora, Miguel A., and Anderson, Wayne F. Sun .  
"A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure".  United Kingdom.  https://doi.org/10.1016/j.jmb.2015.01.009.

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@article{osti_1233990,

  title        = {A Novel Polyamine Allosteric Site of SpeG from Vibrio cholerae Is Revealed by Its Dodecameric Structure},

  author       = {Filippova, Ekaterina V. and Kuhn, Misty L. and Osipiuk, Jerzy and Kiryukhina, Olga and Joachimiak, Andrzej and Ballicora, Miguel A. and Anderson, Wayne F.},

  abstractNote = {Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis.},

  doi          = {10.1016/j.jmb.2015.01.009},

  journal      = {Journal of Molecular Biology},

  number       = 6 PB,

  volume       = 427,

  place        = {United Kingdom},

  year         = {Sun Mar 01 00:00:00 EST 2015},

  month        = {Sun Mar 01 00:00:00 EST 2015}

}

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