Pyrophosphatase is essential for growth of Escherichia coli

Chen, J; Brevet, Annie; Fromant, Michel; Lévêque, F.; Schmitter, Jean‐Marie; Blanquet, Sylvain; Plateau, Pierre

doi:10.1128/jb.172.10.5686-5689.1990

Cited by 191 publications

(162 citation statements)

References 22 publications

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“…PPase is an essential protein, and experiments regulating its intracellular concentration reveal that cellular growth is inhibited when PP i concentrations exceed 2-3 mM (43). Surprisingly, typical PP i concentrations in log-phase E. coli cells are between 0.5 and 1 mM (43,44), well above the concentration at which it fully inhibits tRNA synthetases in vitro (21,45). Perhaps the majority of the PP i is sequestered in an intracellular compartment such that it is unavailable to inhibit aaRSs.…”

Section: Discussionmentioning

confidence: 99%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

124

163

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A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3 -32 P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA Ala . The effect of tRNA Ala identity mutations on both aminoacylation efficiency (kcat͞KM) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the kcat͞KM value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA Ala identity.T wo major approaches have been developed to identify nucleotides required for tRNA recognition by aminoacyl-tRNA (aa-tRNA) synthetases (aaRSs). The first involves introducing a suppressor anticodon into a tRNA of interest and then examining the suppressor activity of the point mutations in vivo (1). The second involves assaying aminoacylation activity of mutant tRNAs made by transcription in vitro (2). Both methods reveal a limited number of nucleotides critical for aminoacylation, the importance of which was confirmed by performing ''swap'' experiments in which the proposed nucleotides were transplanted into the body of another tRNA (2, 3). Subsequent analysis of aminoacylation rate in vitro or sequencing of the reporter proteins in vivo was used to confirm that change in tRNA identity had occurred. These two methods generally agree reasonably well with each other and correlate well with observed base-specific contacts in cocrystal structures of tRNA-aaRS complexes (4-6). However, there are several examples where the conclusions derived from in vitro and in vivo approaches disagree, the most notable of which is the recognition of tRNA Ala by Escherichia coli alanyl-tRNA synthetase (AlaRS). Although no cocrystal structure is available, in vitro experiments with truncated RNA substrates (7, 8) and mutant tRNA Ala (9) as well as identity-swap experiments (10, 11) strongly suggested that the G3⅐U70 pair is the major identity element of tRNA Ala . The deleterious effect of mutations of this base pair significantly exceeds those of other tRNA Ala recognition elements (8, 9, 12-15) and clearly was indicative of the predominant role of this base pair and in particular the exocyclic amino group of G3 in recognition. In contrast, in vivo experiments showed that many of same mutations in the G3⅐U70 base pair have only a moderate effect on the levels of aminoacylated tRNA Ala in E. coli and consequently on the ability of the mutant tRNAs to function in protein synthesis (16,17). A careful analysis of the in vivo experiments indicates that the observed discrepancy was not caused by the technical problems associated with the determining of tRNA aminoacylation levels in vivo, and therefore it was proposed that the discre...

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Section: Discussionmentioning

confidence: 99%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

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

124

163

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“…In addition to fungi (53,77,78), mammals (44,68,69), and invertebrates (75), Fhit homologs can be found in the genomes of microsporidia such as E. cuniculi (83), mycetozoa such as D. discoidium (unpublished), and green plants such as A. thaliana (84). According to the tree of eukaryotic life constructed from protein data (85), unless Fhit is found in plants due to a lateral transfer, the earliest rift that would have preceded appearance of Fhit was the separation of plants from animals, fungi, microsporidia and mycetozoa.…”

Section: Fhit Homologs: Tumor Suppressors and Ap N A Hydrolasesmentioning

confidence: 99%

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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HIT (histidine triad)1 proteins, named for a motif related to the sequence HφHφHφφ, (φ a hydrophobic amino acid) are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5′-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylylsulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding-site. The potential roles of ASW and Hint in avian sexual development are discussed in an accompanying manuscript. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases. Mammalian Hint and GalT Structurally Define the HIT SuperfamilyGalactose-1-phosphate uridylyltransferase (GalT), the second enzyme in the Leloir pathway of galactose utilization (1), was crystallized and its structure determined to understand the mechanistic basis for transferring UMP between glucose-1-phosphate and galactose-1-phosphate (2-4). Histidine triad nucleotide-binding protein (Hint), purified as a purine nucleoside/nucleotide-binding protein from rabbit heart cytosol (5) and shown to have a widely conserved sequence (6), was crystallized and its structure determined to establish whether the conserved sequence formed the basis for a new mode of nucleotide-binding (7). GalT (2) and Hint are both dimers (8) but the GalT monomer is more than twice the size of the Hint dimer. Though GalT (9) and Hint (6) homologs could be cloned by similarity and the crystal structures (2,8) co-existed in protein databases, mutual similarity was not noted until inspection of the rabbit Hint crystal structure (7) and computational analysis (10) indicated that a GalT monomer and a Hint dimer can be superimposed and that their nucleotide-binding sites retain some of the same residues for binding a nucleoside monophosphate (7) (Figure 1). The level of sequence similarity of Hint and GalT is difficult to distinguish from n...

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“…In many cases, the role of PPiase enzymes is to keep the equilibrium of these biosynthetic reactions on the side of the product by hydrolysis of the coproduct of the anabolic reaction. In keeping with this role, loss-of-function mutations of genes encoding PPiases are not viable in certain prokaryotes (Chen et al, 1990).…”

Section: Role Of Pyrophosphatases In Metabolismmentioning

confidence: 99%

Oil and Protein Accumulation in Developing Seeds Is Influenced by the Expression of a Cytosolic Pyrophosphatase in Arabidopsis

et al. 2012

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This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that genes encoding cytosolic pyrophosphatase enzymes provide novel targets to improve seed composition for plant biotechnology applications.

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Pyrophosphatase is essential for growth of Escherichia coli

Cited by 191 publications

References 22 publications

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Oil and Protein Accumulation in Developing Seeds Is Influenced by the Expression of a Cytosolic Pyrophosphatase in Arabidopsis

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Pyrophosphatase is essential for growth of Escherichia coli

Cited by 191 publications

References 22 publications

Modulation of tRNA Ala identity by inorganic pyrophosphatase

Modulation of tRNA Ala identity by inorganic pyrophosphatase

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Oil and Protein Accumulation in Developing Seeds Is Influenced by the Expression of a Cytosolic Pyrophosphatase in Arabidopsis

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Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase