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J. Biol. Chem., Vol. 281, Issue 29, 20521-20529, July 21, 2006

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Structure of Pyrimidine 5'-Nucleotidase Type 1

INSIGHT INTO MECHANISM OF ACTION AND INHIBITION DURING LEAD POISONING^*

From the Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, Wisconsin 53706-1544

Eukaryotic pyrimidine 5'-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5'-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35Å resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25Å_, 2) phosphoenzyme intermediate analog at 2.30Å_, 3) product-transition complex analog at 2.35Å_, and 4) product complex at 2.1Å resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35Å₎ revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.

Received for publication, March 2, 2006 , and in revised form, May 3, 2006.

The atomic coordinates and structure factors (code 2bdu, 2g06, 2g07, 2g08, 2g09, and 2g0a) 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 NIGMS, National Institutes of Health Grants P50 GM64598 and U54 GM074901 (to J. L. Markley, principal investigator). 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 To whom correspondence should be addressed. Tel.: 608-263-6142; Fax: 608-263-6142; E-mail: phillips{at}biochem.wisc.edu.

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