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J. Biol. Chem., Vol. 280, Issue 23, 22318-22325, June 10, 2005

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Identification of a Subversive Substrate of Trichomonas vaginalis Purine Nucleoside Phosphorylase and the Crystal Structure of the Enzyme-Substrate Complex^*

Yang Zang, Wen-Hu Wang, Shaw-Wen Wu, Steven E. Ealick, and Ching C. Wang¶

From the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301 and the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280

Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2'-deoxyadenosine (F-dAdo) is a "subversive substrate." Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC₅₀ values of 106 and 84 nM, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2'-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 Å. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 Å as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.

Received for publication, February 17, 2005 , and in revised form, March 14, 2005.

The atomic coordinates and structure factors (codes 1Z33, 1Z34, 1Z35, 1Z36, 1Z37, 1Z38, and 1Z39) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants AI-60660 (to C. C. W.) and CA-67763 (to S. E. E.). The work at beam line 8-BM was supported by National Institutes of Health Grant RR15301. 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.

^¶ To whom correspondence should be addressed. Tel.: 415-476-1321; Fax: 415-476-3382; E-mail: ccwang{at}cgl.ucsf.edu.

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