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J. Biol. Chem., Vol. 281, Issue 30, 21410-21421, July 28, 2006

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Structural and Mutational Analysis of Substrate Complexation by Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus^*

Marco Marino¹, Miriam Deuss^¶1, Dmitri I. Svergun^||**, Petr V. Konarev^||**, Reinhard Sterner^¶, and Olga Mayans²

From the Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany, ^¶Institut für Biochemie, Universität zu Köln, Otto-Fischer-Strasse 12-14, D-50674 Köln, Germany, ^||European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany and ^**Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia

The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg²⁺. These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/nucleoside recognition by these enzyme families.

Received for publication, February 14, 2006 , and in revised form, May 10, 2006.

The atomic coordinates and structure factors (codes 2GVQ, 1ZXY, and 1ZYK) 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 Deutsche Forschungsgemeinschaft Grant STE 891/5-1 in the framework of the Priority Program 1170. 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 Methods and additional references.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 41-61-267-2083; Fax: 41-61-267-2109; E-mail: Olga.Mayans{at}unibas.ch.

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