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Title: A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by Its dodecameric structure

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 » 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.« less

Authors:
 [1];  [1];  [2];  [1];  [2];  [3];  [1]
  1. Northwestern Univ., Chicago, IL (United States). Center for Structural Genomics of Infectious Diseases.
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
  3. Loyola Univ. Chicago, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (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 Volume: 427; Journal Issue: PB; Journal ID: ISSN 0022-2836
Publisher:
Elsevier
Country of Publication:
United States
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 States: N. p., 2015. Web. doi:10.1016/j.jmb.2015.01.009.
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 States. doi:10.1016/j.jmb.2015.01.009.
Filippova, Ekaterina V., Kuhn, Misty L., Osipiuk, Jerzy, Kiryukhina, Olga, Joachimiak, Andrzej, Ballicora, Miguel A., and Anderson, Wayne F. Fri . "A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by Its dodecameric structure". United States. doi:10.1016/j.jmb.2015.01.009.
@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 = PB,
volume = 427,
place = {United States},
year = {2015},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1016/j.jmb.2015.01.009

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Cited by: 8 works
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