Investigating the Role of Metal Ions in the Catalytic Mechanism of the Yeast RNA Triphosphatase

Bisaillon, Martin; Bougie, Isabelle

doi:10.1074/jbc.m303007200

Cited by 13 publications

(20 citation statements)

References 31 publications

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“…This is in agreement with the model that has been previously proposed for the S. cerevisiae RNA triphosphatase [32] in which the β strands that constitute the active site of the yeast enzyme are already positioned prior to the binding of the substrate (ATP or RNA). Our spectroscopic data indicate that the binding of the ATP substrate does not lead to significant structural modifications of the protein architecture.…”

Section: Discussionsupporting

confidence: 92%

“…Growing evidence suggests that the enzymatic reaction catalysed by enzymes that belong to the metal-dependent RNA triphosphatase family requires a very precise alignment between the residues in the active site of the protein, the substrate and the essential metal-ion cofactor [32]. For instance, the fact that the RNA triphosphatase activity is supported by magnesium, but not by manganese or cobalt, while the ATPase activity is activated by manganese and cobalt, highlights the delicate differences that exist between the two phosphohydrolase activities.…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

Bougie

Bisaillon

2006

Biochemical Journal

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Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5' end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

Bougie

Bisaillon

2006

Biochemical Journal

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“…Although the enzyme is activated by Mg 2ϩ , Ca 2ϩ inhibits its activity by competition with Mg 2ϩ (18). An investigation of the role of metal ion on the phosphatase activity of Cet1 (30) revealed that the binding of Mn 2ϩ did not trigger a major conformational rearrangement. Instead, it presumably perturbed the alignment of the active site residues that ultimately influence substrate catalysis.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Catalytic Mechanism of Mammalian 25-kDa Thiamine Triphosphatase

Song

Bettendorff

Tonelli

et al. 2008

Journal of Biological Chemistry

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Mammalian soluble thiamine triphosphatase (ThTPase) is a 25-kDa cytosolic enzyme that specifically catalyzes the conversion of thiamine triphosphate (ThTP) to thiamine diphosphate and has an absolute requirement for divalent cations. We have investigated the kinetic properties of recombinant mouse thiamine triphosphatase (mThTPase) and determined its solution structure by NMR spectroscopy. Residues responsible for binding Mg 2؉ and ThTP were determined from NMR titration experiments. The binding of Mg 2؉ induced only a minor local conformational change, whereas ThTP binding was found to cause a more global conformational change. We derived a structural model for the mThTPase⅐ThTP⅐Mg 2؉ ternary complex and concluded from this that whereas free mThTPase has an open cleft fold, the enzyme in the ternary complex adopts a tunnel fold. Our results provide a functional rationale for a number of conserved residues and suggest an essential role for Mg 2؉ in catalysis. We propose a mechanism underlying the high substrate specificity of mThTPase and discuss the possible role of water molecules in enzymatic catalysis.Two classes of enzymes are known to convert thiamine triphosphate (ThTP) 3 to thiamine diphosphate in animal tissues: a membrane-associated enzyme that is yet to be characterized and a soluble thiamine triphosphatase (ThTPase). ThTPase was first identified in rat brain (1) and has been found in most mammalian tissues studied to date (2). Biochemical studies of the purified, homogeneous ThTPase further revealed that the enzyme is a 24 -25-kDa monomer with virtually absolute specificity for ThTP (3, 4). The enzyme has an alkaline pH optimum, and its catalytic activity depends critically on a divalent metal cofactor, such as Mg 2ϩ . ThTPase is thought to regulate the intracellular concentration of ThTP, which is found in most animal cells but at a much lower level than other forms of thiamine (5). Several lines of biochemical evidence have shown that ThTP can phosphorylate proteins in the electric organ of Torpedo marmorata and in rodent brain (6). In addition, ThTP is believed to act as one of the messengers in cell signaling in response to cellular stress (7). Despite the relatively high turnover rate of ThTP in neural cells (8), its level is strictly maintained at a low concentration in vivo, presumably through the activity of ThTPase.The sequence of ThTPase does not closely resemble that of any other protein identified in the mammalian genomes. However, recent bioinformatic analysis has suggested that mammalian 25-kDa ThTPase and adenylate cyclase CyaB from Aeromonas hydrophila define an ancient superfamily of domains, called the CYTH domains, which play a role at the interface of polyphosphate and nucleotide metabolism (9). The structures of several CYTH domains have been determined, including class IV adenylyl cyclase from Yersinia pestis (YpAC4, Protein Data Bank (PDB) 2FJT) (10) and proteins with unknown functions from Pyrococcus furiosus (PDB accession numbers 1YEM and 2DC4), Vibrio parahemeolyticus (P...

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“…Further, Cet1p has a single manganese ion coordinated by three glutamates at the floor of the triphosphate tunnel and sulfate with octahedral geometry (Lima et al 1999). The presence of manganese ion in the tunnel has been implicated in triphosphatase reaction (Bisaillon and Bougie 2003).…”

mentioning

confidence: 99%

A Novel Role for Cet1p mRNA 5′-Triphosphatase in Promoter Proximal Accumulation of RNA Polymerase II in Saccharomyces cerevisiase

et al. 2014

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Yeast mRNA 59-triphosphatase, Cet1p, recognizes phosphorylated-RNA polymerase II as a component of capping machinery via Ceg1p for cotranscriptional formation of mRNA cap structure that recruits cap-binding complex (CBC) and protects mRNA from exonucleases. Here, we show that the accumulation of RNA polymerase II at the promoter proximal site of ADH1 is significantly enhanced in the absence of Cet1p. Similar results are also found at other genes. Cet1p is recruited to the 59 end of the coding sequence, and its absence impairs mRNA capping, and hence CBC recruitment. However, such an impaired recruitment of CBC does not enhance promoter proximal accumulation of RNA polymerase II. Thus, Cet1p specifically lowers the accumulation of RNA polymerase II at the promoter proximal site independently of mRNA cap structure or CBC. Further, we show that Cet1p's N-terminal domain, which is not involved in mRNA capping, decreases promoter proximal accumulation of RNA polymerase II. An accumulation of RNA polymerase II at the promoter proximal site in the absence of Cet1p's N-terminal domain is correlated with reduced transcription. Collectively, our results demonstrate a novel role of Cet1p in regulation of promoter proximal accumulation of RNA polymerase II independently of mRNA capping activity, and hence transcription in vivo.

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Investigating the Role of Metal Ions in the Catalytic Mechanism of the Yeast RNA Triphosphatase

Cited by 13 publications

References 31 publications

Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

Structural Basis for the Catalytic Mechanism of Mammalian 25-kDa Thiamine Triphosphatase

A Novel Role for Cet1p mRNA 5′-Triphosphatase in Promoter Proximal Accumulation of RNA Polymerase II in Saccharomyces cerevisiase

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