Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of Aeromonas hydrophila
10.1128/jb.187.18.6370-6378.2005
Crossref journal-article
American Society for Microbiology
Journal of Bacteriology (235)
Abstract

ABSTRACT Aeromonas hydrophila secretes a number of degradative enzymes and toxins into the external milieu via the type II secretory pathway or secreton. ExeA is an essential component of this system and is necessary for the localization and/or multimerization of the secretin ExeD. ExeA contains two sequence motifs characteristic of the Walker superfamily of ATPases. Previous examination of substitution derivatives altered in these motifs suggested that ATP binding or hydrolysis is required for ExeAB complex formation and subsequent secretion function. To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity and anion-exchange chromatographic techniques. Purified preparations of cytExeA exhibited ATPase activity in the presence of several divalent cations, Mg 2+ being the preferred cation, with an optimum reaction temperature of ∼37 to 42°C and an optimum pH of 7 to 8. cytExeA exhibited an apparent K m for Mg-ATP of 0.22 mM and a V max of 0.72 nmol min −1 mg −1 of protein. cytExeA displayed low specificity for nucleoside triphosphate substrates and was significantly inhibited by F-type ATPase inhibitors. Gel filtration analyses of cytExeA, ExeA, and ExeAB indicated that ExeA dimerizes and forms a very large complex with ExeB. These findings support a model whereby ExeAB utilizes energy derived from ATP hydrolysis to facilitate the correct localization and multimerization of the ExeD secretin.

Bibliography

Schoenhofen, I. C., Li, G., Strozen, T. G., & Howard, S. P. (2005). Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of Aeromonas hydrophila. Journal of Bacteriology, 187(18), 6370–6378.

Authors 4
  1. Ian C. Schoenhofen (first)
  2. Gang Li (additional)
  3. Timothy G. Strozen (additional)
  4. S. Peter Howard (additional)
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Dates
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Created 19 years, 11 months ago (Sept. 2, 2005, 5:57 p.m.)
Deposited 4 years, 1 month ago (July 29, 2021, 1:24 p.m.)
Indexed 1 year ago (Aug. 5, 2024, 10:45 p.m.)
Issued 19 years, 11 months ago (Sept. 15, 2005)
Published 19 years, 11 months ago (Sept. 15, 2005)
Published Print 19 years, 11 months ago (Sept. 15, 2005)
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@article{Schoenhofen_2005, title={Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of Aeromonas hydrophila}, volume={187}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.187.18.6370-6378.2005}, DOI={10.1128/jb.187.18.6370-6378.2005}, number={18}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Schoenhofen, Ian C. and Li, Gang and Strozen, Timothy G. and Howard, S. Peter}, year={2005}, month=sep, pages={6370–6378} }