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Journal of Bacteriology, August 2001, p. 4866-4875, Vol. 183, No. 16
Daniel B. Warnell School of Forest Resources
and Department of Biochemistry and Molecular Biology, University of
Georgia, Athens, Georgia 30602-2152
Received 12 March 2001/Accepted 31 May 2001
A gene (yacK) encoding a putative multicopper
oxidase (MCO) was cloned from Escherichia coli, and the
expressed enzyme was demonstrated to exhibit phenoloxidase and
ferroxidase activities. The purified protein contained six copper atoms
per polypeptide chain and displayed optical and electron paramagnetic
resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong optical
A610 (E610 = 10,890 M
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4866-4875.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Oxidation of Phenolate Siderophores by the
Multicopper Oxidase Encoded by the Escherichia coli
yacK Gene
1 cm
1) and copper stoichiometry were taken
as evidence that, similar to ceruloplasmin, the enzyme likely contains
multiple type 1 copper centers. The addition of copper led to immediate
and reversible changes in the optical and EPR spectra of the protein,
as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of
the enzyme, but the other metals tested had no effect. In the presence
of added copper, the enzyme displayed significant activity against two
of the phenolate siderophores utilized by E. coli for
iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as
3-hydroxyanthranilate, an iron siderophore utilized by
Saccharomyces cerevisiae. Oxidation of enterobactin
produced a colored precipitate suggestive of the polymerization
reactions that characterize microbial melanization processes. As
oxidation should render the phenolate siderophores incapable of binding
iron, yacK MCO activity could influence levels of free
iron in the periplasm in response to copper concentration. This
mechanism may explain, in part, how yacK MCO
moderates the sensitivity of E. coli to copper.
*
Corresponding author. Mailing address: School of Forest
Resources, University of Georgia, Athens, GA 30602-2152. Phone: (706) 542-1710. Fax: (706) 542-8356. E-mail: jeffdean{at}uga.edu.
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