Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily

Keppetipola, Niroshika; Jain, Ruchi; Shuman, Stewart

doi:10.1074/jbc.m611328200

Cited by 28 publications

(84 citation statements)

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“…Purified recombinant CthTTM is a vigorous inorganic tripolyphosphatase that converts PPP i to PP i plus P i in the presence of manganese or magnesium. In fact, CthTTM is 2 orders of magnitude more active in cleaving PPP i than it is in hydrolyzing the ␤-␥ phosphoanhydride of ATP (26). By contrast, the Cet1-like RNA triphosphatases are much more active with ATP than PPP i .…”

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confidence: 94%

“…In the open state, the essential amino acid side chains that comprise the active site are displaced from the catalytically active arrangement seen in the Cet1-Mn 2ϩ /SO 4 2Ϫ complex (2). We speculated previously that binding of the polyanionic substrate to the open conformation of the bacterial/archaeal-type TTM apoenzyme might trigger closure of the tunnel and the formation of a proper Michaelis complex (26). Similar inferences regarding substrate-induced conformational changes have been drawn for mammalian thiamine triphosphatase (25), based on documentation of alternative atomic structures analogous to the open and closed states depicted in Fig.…”

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confidence: 99%

“…Here we focus our attention on Clostridium thermocellum TTM (CthTTM), the first bacterial TTM-type phosphohydro-lase to be subjected to detailed biochemical and structure-function analysis (26). Purified recombinant CthTTM is a vigorous inorganic tripolyphosphatase that converts PPP i to PP i plus P i in the presence of manganese or magnesium.…”

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Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Jain

Shuman

2008

Journal of Biological Chemistry

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Triphosphate tunnel metalloenzymes (TTMs) are a superfamily of phosphotransferases with a distinctive active site located within an eight-stranded ␤ barrel. The best understood family members are the eukaryal RNA triphosphatases, which catalyze the initial step in mRNA capping. The RNA triphosphatases characteristically hydrolyze nucleoside 5-triphosphates in the presence of manganese and are inept at cleaving inorganic tripolyphosphate. We recently identified a TTM protein from the bacterium Clostridium thermocellum (CthTTM) with the opposite substrate preference. Here we report that CthTTM catalyzes hydrolysis of guanosine 5-tetraphosphate to yield GTP and P i (K m ‫؍‬ 70 M, k cat ‫؍‬ 170 s ؊1 ) much more effectively than it converts GTP to GDP and P i (K m ‫؍‬ 70 M, k cat ‫؍‬ 0.3 s ؊1 ), implying that a nucleoside interferes when positioned too close to the tunnel entrance. CthTTM is capable of quantitatively cleaving diadenosine hexaphosphate but has feeble activity with shorter derivatives diadenosine tetraphosphate and diadenosine pentaphosphate. We propose that the tunnel opens to accommodate the dumbbell-shaped diadenosine hexaphosphate and then closes around it to perform catalysis. We find that CthTTM can exhaustively hydrolyze a long-chain inorganic polyphosphate, a molecule that plays important roles in bacterial physiology. CthTTM differs from other known polyphosphatases in that it yields a ϳ2:1 mixture of P i and PP i end products. Bacterial/archaeal TTMs have a C-terminal helix located near the tunnel entrance. Deletion of this helix from CthTTM exerts pleiotropic effects. (i) It suppresses hydrolysis of guanosine 5-tetraphosphate and inorganic PPP i ; (ii) it stimulates NTP hydrolysis; and (iii) it biases the outcome of the longchain polyphosphatase reaction more strongly in favor of P i production. We discuss models for substrate binding in the triphosphate tunnel.The RNA triphosphatase components of the mRNA capping systems of fungi, protozoa, and several DNA viruses are the founding members of the triphosphate tunnel metalloenzyme (TTM) 2 superfamily of phosphohydrolases (1-21). The signature biochemical property of this branch of the TTM superfamily is the ability to hydrolyze NTPs to NDPs and P i in the presence of manganese. The crystal structure of the Saccharomyces cerevisiae RNA triphosphatase Cet1 bound to manganese and sulfate (a proposed mimetic of the product complex with phosphate) revealed what was then a novel tertiary structure in which the active site is situated within a topologically closed tunnel composed of eight antiparallel ␤ strands (2). The Cet1-like TTM fold is conserved in the crystal structure of mimivirus RNA triphosphatase, which has an acetate anion bound in its tunnel (21). A plausible mechanism has been proposed whereby Cet1-like proteins catalyze direct attack of a nucleophilic water on the ␥-phosphorus of NTP or pppRNA substrates (4). The putative water nucleophile is oriented by an essential glutamate contributed by the fifth ␤ strand of the tunnel wall. A pr...

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Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Jain

Shuman

2008

Journal of Biological Chemistry

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“…These enzymes belong to, and define, the triphosphate tunnel metalloenzyme superfamily (Gong et al 2006;Keppetipola et al 2007). They share a tunnel architecture and a mechanism of metal-dependent hydrolysis of the γ phosphate of nucleoside triphosphates and RNA 5 ′ -triphosphate ends.…”

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Fission yeast RNA triphosphatase reads an Spt5 CTD code

Doamekpor¹,

Schwer²,

Sánchez³

et al. 2014

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mRNA capping enzymes are directed to nascent RNA polymerase II (Pol2) transcripts via interactions with the carboxy-terminal domains (CTDs) of Pol2 and transcription elongation factor Spt5. Fission yeast RNA triphosphatase binds to the Spt5 CTD, comprising a tandem repeat of nonapeptide motif TPAWNSGSK. Here we report the crystal structure of a Pct1·Spt5-CTD complex, which revealed two CTD docking sites on the Pct1 homodimer that engage TPAWN segments of the motif. Each Spt5 CTD interface, composed of elements from both subunits of the homodimer, is dominated by van der Waals contacts from Pct1 to the tryptophan of the CTD. The bound CTD adopts a distinctive conformation in which the peptide backbone makes a tight U-turn so that the proline stacks over the tryptophan. We show that Pct1 binding to Spt5 CTD is antagonized by threonine phosphorylation. Our results fortify an emerging concept of an "Spt5 CTD code" in which (i) the Spt5 CTD is structurally plastic and can adopt different conformations that are templated by particular cellular Spt5 CTD receptor proteins; and (ii) threonine phosphorylation of the Spt5 CTD repeat inscribes a binary on-off switch that is read by diverse CTD receptors, each in its own distinctive manner.

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“…Known functions of TTMs include fungal and protozoan RNA triphosphatases (Cet1; Lima et al, 1999;Gong et al, 2006), bacterial adenylate cyclases (CyaB from Aeromonas hydrophila and YpAC-IV from Yersinia pestis; Sismeiro et al, 1998;Gallagher et al, 2006), and mammalian thiamine triphosphatases (Lakaye et al, 2004;Song et al, 2008). More recently, tripolyphosphatase activity was discovered for CthTTM from Clostridium thermocellum and NeuTTM from Nitrosomonas europaea, highlighting the functional diversity of this superfamily (Keppetipola et al, 2007;Delvaux et al, 2011). In some instances, TTM proteins can also possess additional domains, adding further complexity to their range of functions.…”

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Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

et al. 2017

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ORCID IDs: 0000-0003-3889-6183 (W.M.); 0000-0002-3797-4277 (K.Y.).The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2. Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

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Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily

Cited by 28 publications

References 24 publications

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Fission yeast RNA triphosphatase reads an Spt5 CTD code

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

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