Regulation and Mutational Analysis of the HPr Kinase/Phosphorylase from Bacillus subtilis ² Fre ´de ´rique Pompeo, ‡ Yohann Granet, ‡ Jean-Pierre Lavergne, § Christophe Grangeasse, § Sylvie Nessler, | Jean-Michel Jault, ⊥ and Anne Galinier* ,‡ Laboratoire de Chimie Bacte ´ rienne, UPR 9043, IBSM-CNRS, 31 chemin Joseph Aiguier, 13402 Marseille, Institut de Biologie et Chimie des Prote ´ ines, UMR 5086 CNRS-UniVersite ´ Claude Bernard Lyon I, 7 Passage du Vercors, 69367 Lyon, Laboratoire d’Enzymologie et de Biochimie Structurales, UPR 9063 CNRS, ba ˆ t. 34, 91198 Gif-sur-YVette, and BMC/DRDC, UMR 5090 CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble cedex 9,France ReceiVed March 12, 2003; ReVised Manuscript ReceiVed April 17, 2003 ABSTRACT: In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the HPr kinase/phosphorylase (HprK/P). It has recently been shown that HprK/P could catalyze the phosphorylation of the protein HPr by using pyrophosphate (PP i ) as a phosphate donor instead of ATP. Here we showed that, as for ATP, PP i binds to the enzyme with strong positive cooperativity. However, in contrast to ATP, PP i binding does not modify the fluorescence properties of the unique Trp residue of Bacillus subtilis HprK/P. In addition, to understand how two conserved motifs, namely, the P-loop and the specific signature of this family, participate in the three enzymatic activities of HprK/Ps (ATP-kinase, PP i -kinase, and phosphorylase), several site-directed mutants were generated. Whereas the three activities are mediated by the P-loop which is directly involved in the binding of ATP, PP i , or Pi, the signature motif seems to be involved preferentially in the dephosphorylation reaction. On the basis of these results, we propose a model in which the binding of the allosteric activator FBP induces a conformational change of a central loop located above the active site of HprK/P, thereby allowing the ATP binding. However, this conformational change is not required for the binding of PP i . Carbon catabolite repression (CCR) 1 allows bacteria to alter catabolic gene expression in response to the availability of rapidly metabolizable carbon sources. In the low-GC Gram-positive bacteria, Bacillus subtilis, regulation of CCR involves the HprK/P, a bifunctional enzyme catalyzing both the phosphorylation and the dephosphorylation of HPr (histidine-containing protein) and its homologue Crh (catabo- lite repression HPr) at Ser46 (1-3). HprK/P appeared to be unrelated to the classical eukaryotic protein kinases, but the catalytic C-terminal domain contains a P-loop (or Walker A motif) commonly found in other nucleotide-binding proteins (4, 5) and a signature motif found in all HprK/P proteins from different organisms (6). The sequence and structure of HprK/P are restrictedly but significantly homologous with those of another phosphotransferase, the phosphoenolpyru- vate carboxykinase (PEPCK) (7, 8). The X-ray structures of HprK/Ps from different species have been determined [PDB entries 1JB1 (9), 1KO7 (10), 1KKL, 1KKM (11), and 1KNX (12)], showing that they are homohexamers. The catalytic core of the protein forms dimers of trimers surrounded by three dimers of N-terminal domains. The function of the latter remains unknown. The ATP-dependent phosphorylation of HPr or Crh is stimulated by glycolytic intermediates such as fructose 1,6- bisphosphate (FBP). It has been demonstrated that B. subtilis HprK/P is an allosteric enzyme which displays strong positive cooperativity for the binding of its allosteric activa- tor, FBP, as well as the binding of the nucleotide ATP (13). Furthermore, inorganic phosphate (P i ) has been found to be another effector of HprK/P which inhibits the kinase activity and stimulates the dephosphorylation reaction. Indeed, in ViVo, the kinase activity is predominant when high concen- trations of ATP and FBP are present in the cell, whereas the dephosphorylation becomes prevalent when the concentration of P i increases (3). The inhibitory effect of P i was explained by demonstrating the competition between P i and ATP for the same binding site (14) and by the crystal structure of HprK/P from Lactobacillus casei (9) and from Staphylococ- cus xylosus (10) that revealed the presence of one or two P i molecules interacting with the P-loop. Recent experiments showed that P i is not an activator of P-Ser-HPr dephospho- rylation but the substrate of the reaction, which produces HPr and pyrophosphate (PP i )(15). The phosphorylase ² This research was supported by the CNRS, the Universite ´ d’Aix- Marseille II, the Fondation pour la Recherche Me ´dicale, and the ministe `re de la recherche “ACI-jeunes-chercheurs” (to A.G.). * To whom correspondence should be addressed. E-mail: galinier@ ibsm.cnrs-mrs.fr. Phone: 33.4.91.16.45.71. Fax: 33.4.91.71.89.14. ‡ IBSM-CNRS. § UMR 5086 CNRS-Universite ´ Claude Bernard Lyon I. | UPR 9063 CNRS. ⊥ UMR 5090 CEA-Grenoble. 1 Abbreviations: CCR, carbon catabolite repression; Crh, catabolite repression HPr; EDTA, ethylenediaminetetraacetic acid; FBP, fructose 1,6-bisphosphate; AMP-PNP, 5′-adenylyl imidodiphosphate; FRET, fluorescence resonance energy transfer; HPr, histidine-containing protein; HprK/P, HPr kinase/phosphorylase; Mant, 2′(3′)-N-methylan- thraniloyl; PEPCK, phosphoenolpyruvate carboxykinase; PFK, phos- phofructokinase; P i, inorganic phosphate; PPi, pyrophosphate. 6762 Biochemistry 2003, 42, 6762-6771 10.1021/bi034405i CCC: $25.00 © 2003 American Chemical Society Published on Web 05/16/2003