A Specific Inorganic Triphosphatase from Nitrosomonas europaea

Abstract

The CYTH superfamily of proteins is named after its two founding members, the CyaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa thiamine triphosphatase. Because these proteins often form a closed ␤-barrel, they are also referred to as triphosphate tunnel metalloenzymes (TTM). Functionally, they are characterized by their ability to bind triphosphorylated substrates and divalent metal ions. These proteins exist in most organisms and catalyze different reactions depending on their origin. Here we investigate structural and catalytic properties of the recombinant TTM protein from Nitrosomonas europaea (NeuTTM), a 19-kDa protein. Crystallographic data show that it crystallizes as a dimer and that, in contrast to other TTM proteins, it has an open ␤-barrel structure. We demonstrate that NeuTTM is a highly specific inorganic triphosphatase, hydrolyzing tripolyphosphate (PPP i ) with high catalytic efficiency in the presence of Mg 2؉ . These data are supported by native mass spectrometry analysis showing that the enzyme binds PPP i (and Mg-PPP i ) with high affinity (K d < 1.5 M), whereas it has a low affinity for ATP or thiamine triphosphate. In contrast to Aeromonas and Yersinia CyaB proteins, NeuTTM has no adenylyl cyclase activity, but it shares several properties with other enzymes of the CYTH superfamily, e.g. heat stability, alkaline pH optimum, and inhibition by Ca 2؉ and Zn 2؉ ions. We suggest a catalytic mechanism involving a catalytic dyad formed by Lys-52 and Tyr-28. The present data provide the first characterization of a new type of phosphohydrolase (unrelated to pyrophosphatases or exopolyphosphatases), able to hydrolyze inorganic triphosphate with high specificity.A large number of phosphohydrolases are able to hydrolyze triphosphorylated compounds, generally nucleoside triphosphates such as ATP or GTP. However, in 2002 we achieved the molecular characterization of a mammalian enzyme that specifically hydrolyzed thiamine triphosphate (ThTP), 6 a compound unrelated to nucleotides (1). This 25-kDa soluble thiamine triphosphatase (ThTPase), which is involved in the regulation of cytosolic ThTP concentrations (2), has near absolute specificity for ThTP (it does not hydrolyze nucleotides) and a high catalytic efficiency. No sequence homology with other known mammalian proteins could be found.Shortly thereafter, Iyer and Aravind (3) observed that the catalytic domains of human 25-kDa ThTPase (1) and CyaB adenylyl cyclase (AC2) from Aeromonas hydrophila (4) define a novel superfamily of domains that, according to these authors, should bind "organic phosphates." This superfamily was called "CYTH" (CyaB-thiamine triphosphatase), and the presence of orthologs was demonstrated in all three superkingdoms of life. This suggested that CYTH is an ancient family of proteins and that a representative must have been present in the last universal common ancestor (LUCA) of all extant life forms. It was proposed (3) that this enzymatic domain might play a central role at the interface between nucleotide a...

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

“…Both AtTTM1 and 2 displayed low activity on ATP, ADP, or PPP i substrates, while high affinity was observed for PP i with a K m of 17 mM, which is comparable to the K m of AtTTM3 or NeuTTM for PPP i (43 mM and 21 mM, respectively; Moeder et al, 2013;Delvaux et al, 2011). We tested several biologically relevant diphosphates such as thiamine diphosphate (Jordan, 2007), ADP ribose (Adams-Phillips et al, 2010) and NADH (Ishikawa et al, 2010), all of which were not hydrolyzed by AtTTM1 and AtTTM2 (Ung and Yoshioka, unpublished data).…”

“…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.…”

Arabidopsis Triphosphate Tunnel Metalloenzyme2 Is a Negative Regulator of the Salicylic Acid-Mediated Feedback Amplification Loop for Defense Responses

“…So far, the exact biological function of TTM proteins in plants is not clear. We previously analyzed AtTTM3 and found that it does not display adenylate cyclase activity, despite its annotation, but it does act on PPPi and with lower affinity, nucleotide triphosphates, releasing inorganic phosphate, similar to the TTM proteins from Clostridium thermocellum (CthTTM) and Nitrosomonas europaea (NeuTTM; Keppetipola et al, 2007;Delvaux et al, 2011;Bettendorff and Wins, 2013;Moeder et al, 2013). Additionally, a transfer DNA (T-DNA) insertion knockout (KO) line of AtTTM3 displayed a delay in root growth as well as reduced length and number of lateral roots, suggesting a role for AtTTM3 in root development.…”

Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS

“…Nevertheless, P 3 has never been reported in prokaryotes, in contrast to long-chain polyPs, although it is known as an intermediate in a number of biosynthetic pathways, e.g. as an intermediate of S-adenosylmethionine, and is generated in some enzymic processes (Bettendorff & Wins, 2013;Delvaux et al, 2011). In contrast to this, P 3 has been shown as a major polyP in acidocalcisomes of several parasitic protists (Moreno et al, 2000), the vacuole of yeast (Castro et al, 1995) and the halotolerant microalga Dunaliella (Pick & Weiss, 1991), and the acidocalcisome-like, mitochondrial and nuclear compartments of mammalian cells (Kumble & Kornberg, 1995;Abramov et al, 2007;Müller et al, 2009;Seidlmayer et al, 2012).…”

“…A group of CYTH proteins named triphosphate tunnel metalloenzymes (TTMs) has been found recently in some bacteria (e.g. Clostridium thermocellum, Nitrosomonas europaea) (Keppetipola et al, 2007;Delvaux et al, 2011) and the plant Arabidopsis thaliana (Moeder et al, 2013), and was reported to be composed of highly specific inorganic tripolyphosphatases. However, the specific metabolic roles of TTM proteins and their contribution, together with the more widespread Ppx-GppA phosphatases, to the ubiquitous tripolyphosphatase activity have not been studied so far.…”