A New Family of Phosphotransferases with a P-loop Motif

Galinier, Anne; Lavergne, Jean‐Pierre; Geourjon, Christophe; Fieulaine, Sonia; Nessler, Sylvie; Jault, Jean‐Michel

doi:10.1074/jbc.m109527200

Cited by 27 publications

(40 citation statements)

References 32 publications

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“…Residues 178-179 form the tip of the ␤D-␤E hairpin, where the polypeptide chain forms a turn with an unusual main-chain conformation (12). Catalytic activity is lost after substitution of either aspartate (14). The x-ray structure suggests that Asp-179 is the catalytic base, but the role of Asp-178 is less obvious.…”

Section: Resultsmentioning

confidence: 92%

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X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

Fieulaine

Moréra

Poncet

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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103

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HPr kinase͞phosphorylase (HprK͞P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK͞P with Bacillus subtilis HPr, both at 2.8-Å resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK͞P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions. P rotein phosphorylation plays a central role in signal transduction and cellular regulation (1). In bacteria, Ser͞Thr kinases are involved in the regulation of a large number of catabolic genes optimizing the use of carbon, nitrogen, and sulfur sources (2). Carbon catabolite repression is a response to metabolic and environmental conditions (3, 4), which stimulates the expression of glycolytic enzymes and represses enzymes catalyzing the metabolism of less efficient carbon sources. In Bacillus subtilis, the expression of Ϸ10% of the genome is regulated by carbon catabolite repression (5, 6).In Gram-positive bacteria, the first step of the carbon catabolite repression response is the phosphorylation on residue Ser-46 of the histidine phosphocarrier protein (HPr). HPr can be phosphorylated also on His-15 by the phosphoenolpyruvate: sugar phosphotransferase system, a general system for sugar transport in bacteria. His-15 phosphorylation is phosphoenolpyruvate-dependent, and the phosphate is transferred to the transported carbohydrate (7). In contrast, the serine modification is ATP-dependent and plays a regulatory role. Serinephosphorylated HPr (P-Ser-HPr) interacts with the catabolite control protein A, forming a complex that binds to DNA at the catabolite response elements cre (8, 9). Phosphorylation is achieved by a specific kinase that also is able to dephosphorylate Ser-46 (10).In the accompanying paper (11), we show that the dephosphorylation of Ser-46 involves the phosphorolysis of the phosphoserine rather than its hydrolysis. Inorganic phosphate (P i ) is a substrate and pyrophosphate (PP i ) is a product of this reaction. Thus, w...

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Section: Resultsmentioning

confidence: 92%

“…PCK and HprK͞P have similar ATP-binding domains and active sites (14,15). All residues of the HprK͞P active site mentioned above have their equivalent in PCK.…”

Section: Resultsmentioning

confidence: 99%

X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

Fieulaine

Moréra

Poncet

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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103

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“…B. subtilis HprK/P was reported to form hexamers at a neutral pH but was reported to form monomers and dimers at pH 9.5 (697). HprK/Ps do not exhibit similarity to eukaryotic protein kinases (312,866) or P-protein phosphatases (43,805) but resemble PEP carboxykinase and nucleosidediphosphate kinases (229,252,748). A P loop (or Walker motif A [GXXGXGKS]) that is usually located between amino acids 150 and 170 serves as a nucleotide binding site.…”

Section: Characteristics Of Atp-dependent Hpr Phosphorylationmentioning

confidence: 99%

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Deutscher

Francke

Postma

2006

Microbiol Mol Biol Rev

1,187

769

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SUMMARY The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

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“…Nucleotide Binding Analysis-We superimposed each subunit of ⌬HPrK/P-V267F with the nucleotide-binding domain of the ATP-bound PEP carboxykinase (PDB code 1AYL) (32), the closest structural homologue of HPrK/P (14). Contrary to the situation described for wild type L. casei ⌬HPrK/P (9), the conformation changes observed with the ⌬HPrK/P-V267F mutant allowed us to directly model bound ATP without any clashes between the base part of the nucleotide and the central loop of the enzyme (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The HPrK/P fold is therefore different from that of eukaryotic protein kinases but resembles small molecule kinases from the P-loop-containing protein family (13). Thus, HPrK/P represents the first member of a novel family of protein kinases (14).…”

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confidence: 99%

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

Chaptal

Vincent

Gueguen-Chaignon

et al. 2007

Journal of Biological Chemistry

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The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phosphocarrier 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 2.6 Å 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.In Gram-positive bacteria, HPr kinase/phosphorylase (HPrK/P) 6 is the central regulator of carbon catabolite repression, the paradigm of signal transduction (1). In response to certain metabolites that change their concentration in the presence of rapidly metabolizable carbon sources, HPrK/P either phosphorylates or dephosphorylates Ser-46 of the histidine-containing protein HPr. In Gram-positive bacteria, Ser(P)-46-HPr functions as co-repressor for carbon catabolite repression by interacting with the transcriptional regulator CcpA (catabolite control protein A) that regulates the expression of many genes (2, 3) (for a review, see Ref. 4). In the Gram-negative Neisseria meningitidis devoid of CcpA-mediated carbon catabolite repression, Ser(P)-46-HPr is involved in the cell adhesion process (5), suggesting that HPrK/P might constitute a potential target for new antimicrobial agents in pathogenic bacteria. Finally, the formation of Ser(P)-46-HPr inhibits PrfA, a transcription activator of several virulence genes in Listeria monocytogenes (6).HPrK/P was first identified as a serine protein kinase, and this activity is enhanced in the presence of glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and inhibited in the presence of inorganic phosphate (P i ) (7). Dephosphorylation of Ser(P)-46-HPr by HPrK/P was assumed to be hydrolytic. However, P i was also reported to stimulate the HPrK/P-c...

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A New Family of Phosphotransferases with a P-loop Motif

Cited by 27 publications

References 32 publications

X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

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

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