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J. Biol. Chem., Vol. 280, Issue 12, 11387-11394, March 25, 2005

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The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC^*

Mehmet Berkmen, Dana Boyd, and Jon Beckwith

From the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.

Received for publication, October 18, 2004 , and in revised form, January 10, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a grant from the Austrian Science Fund Erwin Schrödinger Postdoctoral Fellowship J2199-B04 and by National Institutes of Health Grant GM-55090.

To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1920; Fax: 617-738-7664; E-mail: jbeckwith{at}hms.harvard.edu.

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