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J. Biol. Chem., Vol. 282, Issue 48, 34952-34957, November 30, 2007

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Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme^*

Vincent Chaptal¹², Fanny Vincent¹, Virginie Gueguen-Chaignon, Vicente Monedero³, Sandrine Poncet, Josef Deutscher, Sylvie Nessler⁴, and Solange Morera⁵

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur Yvette, France and Microbiologie et Génétique Moléculaire, CNRS/INRA/AgroParisTech, 78850 Thiverval-Grignon, France

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6Å resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.

Received for publication, July 20, 2007 , and in revised form, September 5, 2007.

The atomic coordinates and structure factors (code 2QMH) 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 Agence Nationale de la Recherche, Association pour la Recherche sur le Cancer, CNRS, Institut National de la Recherche Agronomique, and AgroParisTech. 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 Both authors contributed equally to this work.

² Present address: Dept. of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1751.

³ Present address: Laboratorio de Bacterias Lácticas y Probióticos, Instituto de Agroquimica y Tecnologia de Alimentos-Consejo Superior de Investigaciones Cientificas, P. O. Box 73, 46100 Burjassot, Valencia, Spain.

⁴ To whom correspondence may be addressed. Tel.: 33-1-69-82-34-59; Fax: 33-1-69-82-31-29; E-mail: nessler{at}lebs.cnrs-gif.fr. ⁵ To whom correspondence may be addressed. E-mail: morera{at}lebs.cnrs-gif.fr.

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