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J. Biol. Chem., Vol. 282, Issue 44, 32501-32510, November 2, 2007

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Overexpression of a Zn²⁺-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation^*

From the Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Paul D. Coverdell Biomedical and Health Sciences Center, Athens, Georgia 30602

We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn²⁺, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5'-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation.

Received for publication, June 12, 2007 , and in revised form, August 28, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) AF545106 [GenBank] and AY178275 [GenBank] .

^* This work was supported in part by National Institutes of Health Grant AI68647 (to R. D.). 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.

¹ Present address: Unidad de Investigación, Hospital Universitario Puerta del Mar, Cádiz 11009, Spain.

² Present address: Dept. of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

³ Present address: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941, Brazil.

⁴ Supported by a training grant from the Ellison Medical Foundation to the Center for Tropical and Emerging Global Diseases.

⁵ Supported by a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil).

⁶ Present address: Dept. of Pathobiology and Medical Scholar Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

⁷ To whom correspondence should be addressed: Center for Tropical and Emerging Global Diseases and Dept. of Cellular Biology, 500 D.W. Brooks Dr., Paul D. Coverdell Center, University of Georgia, Athens, GA 30602. Tel.: 706-542-8104; Fax: 706-542-3582; E-mail: rdocampo{at}cb.uga.edu.

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