The <i>hprK</i> gene of <i>Enterococcus faecalis</i> encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

Kravanja, M.; Engelmann, Roswitha; Dossonnet, Valérie; Blüggel, Martin; Meyer, Helmut E.; Frank, Rainer; Galinier, Anne; Deutscher, Josef; Schnell, Norbert; Hengstenberg, Wolfgang

doi:10.1046/j.1365-2958.1999.01146.x

Cited by 124 publications

(163 citation statements)

References 28 publications

(45 reference statements)

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“…Thus, we now call the bifunctional enzyme HPr kinase͞ phosphorylase (HprK͞P). The kinase activity is activated by fructose-1,6-bisphosphate and inhibited by P i (10). In starved cells, where the P i concentration is high and that of fructose-1,6-bisphosphate is low, HPr is dephosphorylated.…”

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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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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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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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“…Also, this enzyme is bifunctional, capable of a slow dephosphorylation of Ser(P)-HPr or Ser(P)-Crh, and has therefore been called HPr kinase/phosphatase (HprK/P) (9). Concerning its functioning at the molecular level, HprK/P appeared unrelated to the classical eukaryotic protein kinases since it does not possess the typical signatures found in this family (5,6).…”

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A New Family of Phosphotransferases with a P-loop Motif

Galinier

Lavergne

Geourjon

et al. 2002

Journal of Biological Chemistry

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In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the histidine-containing protein kinase/phosphatase (HprK/P). Based either on its primary sequence or on its recently solved three-dimensional structure, no straightforward homology with other known proteins was found. However, we showed here that HprK/P exhibits a restricted homology with an unrelated phosphotransferase, the phosphoenolpyruvate carboxykinase. This includes notably two consecutive Asp residues from the phosphoenolpyruvate carboxykinase active site, whose equivalent residues were mutated in Bacillus subtilis HprK/P. Characterization of the corresponding mutants emphasizes the crucial role of these Asp residues in the HprK/P functioning. Furthermore, superimposition of HprK/P and phosphoenolpyruvate carboxykinase active sites supports the view that both enzymes bear significant resemblance in their overall mechanism of functioning showing that these two enzymes constitute a new family of phosphotransferases.

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“…By contrast, in wild-type cells actively growing on glucose, P~His-HPr is preferentially utilized for glucose phosphorylation through the PTS, and low steady-state levels of P~His-HPr are probably maintained. The apparent low phosphorylation of HPr at serine-46 in the manM mutant could be explained by two facts: (i) P~His-HPr is a poor substrate for HprK/P-catalysed phosphorylation at serine-46 (Deutscher et al, 1984) and (ii) the reduced rate of sugar utilization caused by a defect in PTS transport would not trigger activation of the kinase function of HprK/P (Dossonnet et al, 2000;Kravanja et al, 1999). It seems, therefore, that the pleiotropic effect of a mutation in the PTS Man proteins could, at least partly, be due to diminished phosphorylation of HPr at serine-46.…”

Section: Discussionmentioning

confidence: 99%

“…P~His-HPr, the form involved in sugar transport through the PTS, can also transfer its phosphate to other non-PTS proteins, thereby regulating their functions (Deutscher et al, 2002;Stülke et al, 1998). Furthermore, HPr can be phosphorylated at residue serine-46 by an ATP-dependent metabolite-activated protein kinase (HPrK/P) (Galinier et al, 1998;Kravanja et al, 1999;Reizer et al, 1998). P-Ser-HPr participates in the mechanism of sugar-uptake regulation known as inducer exclusion and in carbon catabolite repression (CCR), acting as a cofactor of the transcriptional regulator CcpA (Deutscher et al, 1995;Schumacher et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Molecular analysis of the glucose-specific phosphoenolpyruvate : sugar phosphotransferase system from Lactobacillus casei and its links with the control of sugar metabolism

et al. 2006

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Lactobacillus casei transports glucose preferentially by a mannose-class phosphoenolpyruvate : sugar phosphotransferase system (PTS). The genomic analysis of L. casei allowed the authors to find a gene cluster (manLMNO) encoding the IIAB (manL), IIC (manM) and IID (manN) proteins of a mannose-class PTS, and a putative 121 aa protein of unknown function (encoded by manO), homologues of which are also present in man clusters that encode glucose/mannose transporters in other Gram-positive bacteria. The L. casei man operon is constitutively expressed into a manLMNO messenger, but an additional manO transcript was also detected. Upstream of the man operon, two genes (upsR and upsA) were found which encode proteins resembling a transcriptional regulator and a membrane protein, respectively. Disruption of either upsR or upsA did not affect manLMNO transcription, and had no effect on glucose uptake. Cells carrying a manO deletion transported glucose at a rate similar to that of the wild-type strain. By contrast, a manM disruption resulted in cells unable to transport glucose by the PTS, thus confirming the functional role of the man genes. In addition, the manM mutant exhibited neither inducer exclusion of maltose nor glucose repression. This result confirms the need for glucose transport through the PTS to trigger these regulatory processes in L. casei.

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The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

Cited by 124 publications

References 28 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

A New Family of Phosphotransferases with a P-loop Motif

Molecular analysis of the glucose-specific phosphoenolpyruvate : sugar phosphotransferase system from Lactobacillus casei and its links with the control of sugar metabolism

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