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J. Biol. Chem., Vol. 280, Issue 5, 3376-3381, February 4, 2005

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Altered Oxyanion Selectivity in Mutants of UhpT, the P_i -linked Sugar Phosphate Carrier of Escherichia coli^*

Jason A. Hall and Peter C. Maloney

From the Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21205

In Escherichia coli, the UhpT transporter catalyzes the electroneutral accumulation of sugar 6-phosphate by exchange with internal inorganic phosphate (P_i). The substrate specificity of UhpT is regulated at least in part by constituents of an Asp³⁸⁸-Lys³⁹¹ intrahelical salt bridge, and mutations that remove one but not both of these residues alter UhpT preference for organophosphate substrates. Using site-directed mutagenesis, we examined the role played by these two positions in the selection of the oxyanion countersubstrate. We show that derivatives having aliphatic or polar residues at positions 388 and 391 are gain-of-function mutants capable of transporting SO₄ as well as P_i. These oxyanions share similar structures but differ significantly in the presence of a proton(s) on P_i. Our findings therefore lead us to suggest that the Asp³⁸⁸-Lys³⁹¹ ion pair acts normally as a filter that prevents substrates lacking a proton that can be donated from occupying the UhpT active site.

Received for publication, August 30, 2004 , and in revised form, November 9, 2004.

^* This work was supported by National Institutes of Health Grant GM24195. 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.

Present address: Dept. of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0647.

To whom correspondence should be addressed: Dept. of Physiology, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-955-8325; Fax: 410-955-4438; E-mail: pmaloney{at}jhmi.edu.

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