Showing papers by "Anne Galinier published in 2002"

Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life?

Abstract: In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes (≈10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the β phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho–phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations (≈6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.

Carbohydrate Uptake and Metabolism

Abstract: The genes encoding the polysaccharide-hydrolyzing enzymes are often organized in an operon or regulon together with genes that encode the enzymes catalyzing the uptake of the extracellular hydrolytic products and the first intracellular steps in their catabolism. The Bacillus subtilis genome encodes about 23 secondary and 11 ATP-binding cassette (ABC) carbohydrate transporters. The major facilitator superfamily (MFS) comprises eight B. subtilis proteins of unknown carbohydrate specificity exhibiting significant similarity to the GalP/XylE subfamily. The genes encoding the polysaccharide-hydrolyzing enzymes are often organized in an operon or regulon together with genes that encode the enzymes catalyzing the uptake of the extracellular hydrolytic products and the first intracellular steps in their catabolism. Glycolysis is one of the most conserved metabolic pathways in living organisms. To conserve cellular resources, expression of most of the hundreds of carbohydrate catabolism genes is induced only when the corresponding carbohydrate is present in the growth medium. Expression of antiterminator-controlled genes or operons usually occurs from a constitutive promoter; transcription stops at a terminator located in the leader region of these genes and operons, providing very short transcripts. The carbohydrate transport systems operative in gram-positive and gram-negative bacteria are very similar and most likely developed early in evolution.

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

Abstract: HPr kinase/phosphorylase (HprK/P) 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 HprK/P with Bacillus subtilis HPr, both at 2.8-A 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 HprK/P 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.

Insights into the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role of cations and phosphate.

Abstract: In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase (HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 of either HPr (histidine-containing protein) or Crh (catabolite repression HPr). By using a surface plasmon resonance approach, it was shown here that the presence of magnesium is a prerequisite for the interaction of HprK/P with either HPr or Crh. HprK/P binds both protein substrates with a similar affinity (K(D) of about 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate. In addition, the specificity and the concentration of the cation required for the binding of protein substrates are different from that exhibited by the cation-binding site involved in the nucleotide binding, suggesting the presence of two cation-binding sites on HprK/P. The effects of phosphate on enzymatic activities of HprK/P were also investigated. Phosphate was able to unmask the phosphatase activity, especially in the presence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinase activity of HprK/P. An apparent competition between phosphate and a fluorescent analogue of nucleotide led to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of the enzyme.

Dosage simple et rapide de la leucine par CLHP. Intérêt dans l’épuration extrarénale des leucinoses

Abstract: La leucinose ou maladie des urines a odeur de sirop d’erable (connue egalement sous le nom de cetonurie a chaine ramifiee) est secondaire a une anomalie du metabolisme des acides amines ramifies (leucine, isoleucine et valine). Sa frequence est d’environ 1 pour 200 000 naissances [1]. Decrite des 1954 par Menkes et al., cette erreur [...]