title: Purification and characterization of the N-terminal domain of ExeA: a novel ATPase involved in the type II secretion pathway of Aeromonas hydrophila.
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, Mg2+ being the preferred cation, with an optimum reaction temperature of approximately 37 to 42 degrees 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.
authors.affiliation: National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Schoenhofen
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Strozen
authors.forename: S Peter
authors.initials: SP
authors.lastname: Howard
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D016980:Aeromonas hydrophila / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001692:Biological Transport; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C090402:ExeA protein, Aeromonas hydrophila; D026901:Membrane Transport Proteins; D000251:Adenosine Triphosphatases