Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase

Delbaere, L. T. J.; Sudom, Athena; Prasad, L. Guru; Leduc, Y.A.; Goldie, Hughes

doi:10.1016/j.bbapap.2003.11.030

Cited by 41 publications

(34 citation statements)

References 50 publications

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“…Malate metabolizes to OAA (Fig. 1a) and OAA converts to phosphoenolpyruvate (Delbaere et al, 2004). We confirmed that the V. cholerae genome has this enzyme, the phosphoenolpyruvate carboxykinase (VC2738).…”

Section: Discussionsupporting

confidence: 61%

Central metabolism controls transcription of a virulence gene regulator in Vibrio cholerae

et al. 2013

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ToxT is the central regulatory protein involved in activation of the main virulence genes in Vibrio cholerae. We have identified transposon insertions in central metabolism genes, whose disruption increases toxT transcription. These disrupted genes encode the primary respiration-linked sodium pump (NADH : ubiquinone oxidoreductase or NQR) and certain tricarboxylic acid (TCA) cycle enzymes. Observations made following stimulation of respiration in the nqr mutant or chemical inhibition of NQR activity in the TCA cycle mutants led to the hypothesis that NQR affects toxT transcription via the TCA cycle. That toxT transcription increased when the growth medium was supplemented with citrate, but decreased with oxaloacetate, focused our attention on the TCA cycle substrate acetyl-CoA and its non-TCA cycle metabolism. Indeed, both the nqr and the TCA cycle mutants increased acetate excretion. A similar correlation between acetate excretion and toxT transcription was observed in a tolC mutant and upon amino acid (NRES) supplementation. As acetate and its tendency to decrease pH exerted no strong effect on toxT transcription, and because disruption of the major acetate excretion pathway increased toxT transcription, we propose that toxT transcription is regulated by either acetyl-CoA or some close derivative. INTRODUCTIONCholera is a severe waterborne diarrhoeal disease that remains a public health concern in many developing countries. The causative agent of cholera is Vibrio cholerae, a Gram-negative bacterium. The two main essential virulence factors of V. cholerae are toxin-coregulated pilus (TCP) and cholera toxin (CT). TCP is a type IV pilus that is required for colonization of the small intestine. CT is a potent enterotoxin responsible for inducing most of the cholera symptoms. The genes that encode TCP, CT and many other virulence determinants comprise a network of genes called the ToxT regulon, whose expression is modulated by a hierarchy of transcriptional regulators (Matson et al., 2007). These regulators include the transcription factors AphA and AphB, which positively regulate transcription of tcpPH. The membrane-bound protein complex TcpPH works with the membrane-bound protein complex ToxRS to activate transcription of toxT, which encodes an AraC-type transcriptional factor (Matson et al., 2007). While it is ToxT that directly activates the genes that encode TCP, CT and the rest of the ToxT regulon, additional positive regulators have been reported. In contrast, our knowledge concerning factors that negatively regulate the ToxT regulon (reviewed by Matson et al., 2007) is restricted to the nucleoid-associated protein H-NS, which represses transcription of toxT, tcpA and ctxAB (Nye et al., 2000;Stonehouse et al., 2011), and the cAMP-receptor protein (CRP), which is reported to negatively regulate tcpPH (Kovacikova & Skorupski, 2001).In a previous attempt to identify negative regulators of toxT transcription, we found that elevated toxT transcription results from loss of the primary respiration-linked sodium pu...

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Section: Discussionsupporting

confidence: 61%

Central metabolism controls transcription of a virulence gene regulator in Vibrio cholerae

et al. 2013

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“…Our results suggest that acetylation of Pck1p increases its enzymatic activity through mechanisms other than regulating macromolecular abundance, subcellular localization or quaternary structure of the protein. Based on the crystal structure of the Escherichia coli ortholog (Delbaere et al, 2004), which shares high level of amino acid sequence homology with yeast Pck1p (Figure S19), acetylation at K514 might cause a conformational change of the C-terminal mononucleotide-binding domain in favor of substrate binding or catalysis of the phosphoryl-transfer reaction. Despite sharing minimal primary structural identity with yeast Pck1p (Ravanal et al, 2003) (Figure S17), we found that human Pck1 is also activated by TIP60 -dependent acetylation.…”

Section: Discussionmentioning

confidence: 99%

Protein Acetylation Microarray Reveals that NuA4 Controls Key Metabolic Target Regulating Gluconeogenesis

Yang

Zhang

et al. 2009

Cell

289

333

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SUMMARY Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys 19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys 514 was crucial for enzymatic activity and the ability of yeast cells to grow on non-fermentable carbon sources. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production was dependent on TIP60, the human homolog of ESA1. Our results demonstrate a novel regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.

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“…However, further study of highly charged phosphate esters such as nucleoside triphosphates is warranted to further explore this issue. For example, it has been suggested from structural data that enzymes can bind ATP with the phosphoryl oxygen atoms in an eclipsed conformation, introducing steric or electrostatic repulsion that may destabilize the bound substrate and thereby lower the reaction barrier (102, 103). …”

Section: Enzymatic Phosphoryl Transfermentioning

confidence: 99%

Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis

Lassila

Zalatan

Herschlag

2011

Annu. Rev. Biochem.

372

662

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Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.

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Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase

Cited by 41 publications

References 50 publications

Central metabolism controls transcription of a virulence gene regulator in Vibrio cholerae

Central metabolism controls transcription of a virulence gene regulator in Vibrio cholerae

Protein Acetylation Microarray Reveals that NuA4 Controls Key Metabolic Target Regulating Gluconeogenesis

Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis

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