Identification, Purification, and Characterization of an Eukaryotic-like Phosphopantetheine Adenylyltransferase (Coenzyme A Biosynthetic Pathway) in the Hyperthermophilic Archaeon Pyrococcus abyssi

Armengaud, Jean; Fernandez, Bernard; Chaumont, Valérie; Rollin-Genetet, Françoise; Finet, Stéphanie; Marchetti, Charles; Myllykallio, Hannu; Vidaud, Claude; Pellequer, Jean‐Luc; Gribaldo, Simonetta; Forterre, Patrick; Gans, Pierre

doi:10.1074/jbc.m301891200

Cited by 27 publications

(23 citation statements)

References 35 publications

(58 reference statements)

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“…A number of genes that display similarity to bacterial/eukaryotic genes are present on the archaeal genomes, including those encoding PPCS, PPCDC, and PPAT. Among these, the PPCS and PPCDC from Methanocaldococcus jannaschii (13) and the PPAT from Pyrococcus abyssi (1,17) have been biochemically characterized. A striking observation was that almost all of the archaeal genomes did not harbor genes corresponding to PS and PanK.…”

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confidence: 99%

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

et al. 2012

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Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4=-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable k cat /K m values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg 2؉ , respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.

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confidence: 99%

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

et al. 2012

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“…In order to detect possible adenosine nucleotide products of MM2281, standard assays containing MM2281 alone or together with E. coli PANK were carried out as described above, except that [3][4][5][6][7][8][9][10][11][12][13][14] C]b-alanine was omitted. The reaction was quenched after 180 min, and the products were cochromatographed with authentic ATP, ADP and AMP standards (Sigma).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…We previously used comparative genomics to reconstruct the universal CoA biosynthetic pathway in the Bacteria, Eukaryota, and Archaea [10]. Archaeal genes for the ultimate four steps can be identified by homology in all archaeal genomes, and experimental confirmation of this assignment is available for three of these steps [11,12]. In addition, bacterial-type genes for the first two steps are obvious in a number of the nonmethanogenic Archaea.…”

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A novel isoform of pantothenate synthetase in the Archaea

2008

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Pantothenate is the essential precursor to CoA, which is of central importance for all parts of metabolism. This is shown by the fact that more than 400 enzymecatalyzed reactions are known to involve CoA (KEGG database [1]). Many more enzymes utilize acylated forms of CoA or require the CoA-derived phosphopantetheine as a prosthetic group. Typically, plants, fungi and microorganisms are able to synthesize pantothenate de novo, whereas animals rely on pantothenate in their diet.Pantothenate synthetase (PS) catalyzes the last step in the biosynthesis of pantothenic acid, also known as vitamin B 5 . The enzyme (EC 6.3.2.1) has been extensively studied in Escherichia coli [2,3], Mycobacterium tuberculosis [4,5], and Arabidopsis thaliana [6], and is highly conserved in the Bacteria and Eukaryota. Bacterial PS (Eqn 1) generates pantothenate from pantoate and b-alanine. It is an AMP-forming synthetase that proceeds via an acyl-adenylate intermediate and belongs to the HIGH superfamily of nucleotidyltransferases [3].

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“…Large scale liquid cultures were set up at 30°C and induced with 1 mM isopropyl-␥-D-thiogalactopyranoside as described earlier (13). Cells (63 g of wet material) were resuspended in 300 ml of cold 50 mM Tris/HCl buffer (pH 8.3 at 20°C) and disrupted by means of a BasicZ cell disrupter (Constant Systems Ltd.) operated at 900 bars.…”

Section: Construction Of An N-terminal Hismentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. abyssi GE5 PAB0944 gene (COG1019) corresponds to a single domain polypeptide involved in the fourth step of coenzyme A biosynthesis (phosphopantetheine adenylyltransferase), whereas its human ortholog is fused to another domain, dephosphocoenzyme A kinase, which is responsible for catalyzing the last step of the coenzyme A biosynthetic pathway (13). All of these examples clearly indicate that PACE proteins have fundamental cellular functions and that several of them are obviously related to RNA metabolism.…”

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N2-Methylation of Guanosine at Position 10 in tRNA Is Catalyzed by a THUMP Domain-containing, S-Adenosylmethionine-dependent Methyltransferase, Conserved in Archaea and Eukaryota

Armengaud

Urbonavičius

Fernandez

et al. 2004

Journal of Biological Chemistry

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In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed Pab Trm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N 2 ) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N 2 -monomethyl (m 2 G) or N 2 -dimethylguanosine (m 2 2 G). Interestingly, Pab Trm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus ( Pfu Trm-G26, also known as Pfu Trm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life.

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Identification, Purification, and Characterization of an Eukaryotic-like Phosphopantetheine Adenylyltransferase (Coenzyme A Biosynthetic Pathway) in the Hyperthermophilic Archaeon Pyrococcus abyssi

Cited by 27 publications

References 35 publications

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

A novel isoform of pantothenate synthetase in the Archaea

N2-Methylation of Guanosine at Position 10 in tRNA Is Catalyzed by a THUMP Domain-containing, S-Adenosylmethionine-dependent Methyltransferase, Conserved in Archaea and Eukaryota

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