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J. Biol. Chem., Vol. 283, Issue 34, 23295-23304, August 22, 2008

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Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases^*

Ileana Ramazzina¹, Laura Cendron, Claudia Folli, Rodolfo Berni, Daniela Monteverdi^¶, Giuseppe Zanotti, and Riccardo Percudani²

From the Departments of Biochemistry and Molecular Biology and ^¶Mathematics, University of Parma, 43100, Parma, Italy and the Department of Chemistry, University of Padua, 35131 Padua, Italy

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase β/ barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution.

Received for publication, February 13, 2008 , and in revised form, May 7, 2008.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) EU293536 [GenBank] .

The atomic coordinates and structure factors (codes 3CL6, 3CL7, and 3CL8) 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 the Universities of Parma and Padua. 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 Table S1.

¹ Supported by a fellowship from SARCE SpA.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Parco area delle Scienze, University of Parma, 43100. Tel.: 39-0521-905140; Fax: 39-0521-905151; E-mail: riccardo.percudani{at}unipr.it.

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