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J. Biol. Chem., Vol. 283, Issue 2, 840-848, January 11, 2008

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Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant^*

Lluis Masip¹, Daniel Klein-Marcuschamer², Shu Quan, James C. A. Bardwell, and George Georgiou^¶||**3

From the Departments of Chemical Engineering ^¶Biomedical Engineering, and ^||Molecular Genetics and Microbiology and the ^**Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712 and the Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109

Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA^– cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA^– cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.

Received for publication, June 22, 2007 , and in revised form, October 24, 2007.

^* This work was supported in part by National Institutes of Health Grants GM 55090 and GM069872 (to G. G.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

¹ Present address: Dept. of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.

² Supported in part by an REU Grant from the National Science Foundation. Present address: Dept. of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02319.

³ To whom correspondence should be addressed. E-mail: gg{at}che.utexas.edu.

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