Quantification of the Influence of HPrSer46P on CcpA–cre Interaction

Aung‐Hilbrich, Lwin Mar; Seidel, Gerald; Wagner, Andrea; Hillen, Wolfgang

doi:10.1016/s0022-2836(02)00245-0

Cited by 36 publications

(37 citation statements)

References 33 publications

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“…Mutations at Ser-46 also affect the PEP-dependent, EIcatalyzed phosphorylation at His-15. In a mutant complementation assay carried out with rate-limiting amounts of B. subtilis HPr (20 M), the phosphoryl carrier activity was lowered to 35,30, and 20% when Ser46Ala, Ser46Tyr, or Ser46Thr HPr, respectively, was used in place of wild-type HPr (206) and was lowered to 10% when Ser46Asp HPr was used (722). Phosphorylation of HPr at Ser-46 lowered the rate of PEP-dependent phosphorylation by 2 orders of magnitude (177).…”

Section: Characteristics Of Atp-dependent Hpr Phosphorylationmentioning

confidence: 99%

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

Deutscher

Francke

Postma

2006

Microbiol Mol Biol Rev

1,162

752

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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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Section: Characteristics Of Atp-dependent Hpr Phosphorylationmentioning

confidence: 99%

“…Similar results were obtained with cre's from many other catabolite-repressed or -activated transcription units. In addition, by isolating the DNA:protein complex and separating its components on a denaturing gel, it was demonstrated that P-Ser-HPr and CcpA bind together to cre sites (30).…”

Section: P-ser-hpr Functions As a Catabolite Corepressormentioning

confidence: 99%

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

Deutscher

Francke

Postma

2006

Microbiol Mol Biol Rev

1,162

752

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“…CcpA cannot bind HPr-like proteins at physiologically relevant concentrations unless they are phosphorylated on residue Ser 46 (6,26,34). Such phosphorylation of Ser 46 increases the binding affinity of CcpA for cre DNA, indicating the critical nature of this interaction in both binding specificity and affinity (26,34). In addition, several hydrophobic residues on ␣2 interact with hydrophobic residues on CcpA helix IX to form a water-free interface.…”

Section: Ccpa-crh-ser 46 -P Interactions-eachmentioning

confidence: 99%

Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation

Schumacher

Seidel

Hillen

et al. 2006

Journal of Biological Chemistry

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In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser 46 -P or Crh-Ser 46 -P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser 46 -P more weakly than HPr-Ser 46 -P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser 46 -P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser 46 -P)-DNA complex. The structure reveals that Crh-Ser 46 -P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser 46 -P. The reduced affinity of Crh-Ser 46 -P for CcpA as compared with that of HPr-Ser 46 P is explained by weaker CrhSer 46 -P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix ␣2 of the second contact region (contact region II) of Crh-Ser 46 -P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA. Carbon catabolite repression/regulation (CCR)2 is a global regulatory mechanism utilized by bacteria to select, out of a mixture of compounds, the carbon source providing the optimal growth advantage (1-3). CCR is mediated largely at the level of transcription. The master transcriptional regulator of CCR in bacilli and other Gram-positive bacteria with low GC content is the catabolite control protein A (CcpA) (4 -10). CcpA binds to catabolite responsive elements (cre) to mediate its effect (11,12). Approximately 10% of the Bacillus subtilis genome is under regulation by CcpA, underscoring its vital metabolic role (13).CcpA is a member of the LacI-GalR family of transcription regulators (14). LacI-GalR proteins contain a 60-residue N-terminal DNA binding domain that connects to a larger C-terminal domain. The C-terminal domain consists of N-and C-subdomains connected by a hinge region. (6, 24 -26). The recent structure of the CcpA-(HPr-Ser 46 -P)-cre revealed the mechanism by which HPr-Ser 46 -P functions as a corepressor for CcpA (27). Strikingly, this DNA binding activation mechanism is different from those of PurR and LacI in that CcpA utilizes a two-component phosphoprotein that is induced and stabilized by closure of its N-and C-subdomains. This mechanism involves both a rotation of CcpA subdomains as well as a relocation of the key residue Thr 61 , which is located at the interface of the DNAbinding and corepressor-binding domains. The repositioning of this residue leads to a juxtaposition of the DNA-binding domains to permit hinge helix formation in the presence of cognate DNA (15, 16).In addition to HPr, a structural and functional homologue, Crh (for catabolite repression HPr) has been identified, but only in bacilli (28). Crh...

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“…As in other low-G+C Gram-positive bacteria, CCR takes place through the binding of the transcriptional repressor CcpA (Miwa et al, 1994;Hueck et al, 1995;Egeter & Brückner, 1996;Lokman et al, 1997;Leboeuf et al, 2000) to an operator sequence called cre (catabolite responsive element) (Fujita et al, 1995;Aung-Hilbrich et al, 2002). CcpA binding to cre sequences is markedly enhanced by its co-repressor, the Ser-46 phosphorylated HPr protein (P-Ser-HPr), a key component of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) Jones et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Complementation of a ΔccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains

et al. 2004

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In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA-and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features. INTRODUCTIONIn the industrially relevant lactic acid bacterium Lactobacillus casei, the preferential utilization of carbon sources is controlled by the mechanism of carbon catabolite repression (CCR) (Monedero et al., 1997). As in other low-G+C Gram-positive bacteria, CCR takes place through the binding of the transcriptional repressor CcpA (Miwa et al., 1994;Hueck et al., 1995; Egeter & Brückner, 1996;Lokman et al., 1997;Leboeuf et al., 2000) to an operator sequence called cre (catabolite responsive element) (Fujita et al., 1995;Aung-Hilbrich et al., 2002). CcpA binding to cre sequences is markedly enhanced by its co-repressor, the Ser-46 phosphorylated HPr protein (P-Ser-HPr), a key component of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) Jones et al., 1997). This links CCR to the sugar transport process via the PTS (Brückner & Titgemeyer, 2002). Some small molecules, such as fructose 1,6-bisphosphate, glucose 6-phosphate or NADP, can also influence the interaction between CcpA and cre (Gosseringer et al., 1997;Kim et al., 1998). Therefore, the major role attributed to CcpA was related to CCR in the presence of rapidly metabolizable carbon sources.CcpA inactivation pleiotropically affects the expression of approximately 8 % of the genes in Bacillus subtilis (Moreno et al., 2001). Genes of very important metabolic pathways are regulated by CcpA, such as citB and citZ contributing to the Krebs cycle (Kim et al., 2002), gltAB for ammonium assimilation (Faires et al., 1999) or the ilv-leu operon for branched-chain amino-acid biosynthesis (Ludwig et al., 2002). Other studies revealed that CcpA in Gram-positive bacteria also regulates carbon catabolite activation (CCA) of acetoin secretion, acetate biosynthesis genes in B. subtilis (Turinsky et al., 2000) and glycolytic genes in B. subtilis, Lactococcus lact...

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Quantification of the Influence of HPrSer46P on CcpA–cre Interaction

Cited by 36 publications

References 33 publications

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

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

Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation

Complementation of a ΔccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains

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