Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Abstract

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...

A Specific Inorganic Triphosphatase from Nitrosomonas europaea

“…This is all the more plausible as k cat was even higher for the untagged enzyme (up to 7900 s Ϫ1 at 50°C and pH 9.7, see Table 2). This is 2 orders of magnitude higher than k cat values obtained for CthTTM with its best substrates, PPP i and Gp 4 (12,13). This raises the possibility that in N. europaea the physiological function of NeuTTM might be the degradation of PPP i .…”

“…In bacteria, two CYTH orthologs were shown to have an adenylyl cyclase activity (4,11,27), but under physiological conditions this activity is low, and its biological significance is not established. CthTTM, the ortholog from C. thermocellum hydrolyzes PPP i with a relatively good turnover (k cat ϭ 3 s Ϫ1 ), but it also hydrolyzes other substrates such as Gp 4 (k cat ϭ 96 s Ϫ1 ) and, to a lesser extent, nucleoside triphosphates and long chain polyphosphates (12,13). Thus, its possible physiological function remains undefined.…”

“…The enzyme, which was characterized by Shuman and co-workers (12,13) in considerable detail had a rather broad substrate specificity. It hydrolyzed guanosine 5Ј-tetraphosphate (Gp 4 ), inorganic triphosphate (tripolyphosphate (PPP i )), and to a lesser extent nucleoside triphosphates and long-chain polyphosphates (12,13).…”

Crystal Structure of Vaccinia Virus mRNA Capping Enzyme Provides Insights into the Mechanism and Evolution of the Capping Apparatus

“…Early studies of the vaccinia TPase revealed what would emerge as the signature biochemical feature of the TTM-type RNA triphosphatases: the ability to hydrolyze NTP to NDP and P i in the presence of Mn 2+ or Co 2+ (Shuman et al, 1980). The yeast Cet1 structure unveiled a then novel tunnel tertiary structure (Lima et al, 1999) that has since been recognized as the founder of a TTM superfamily that extends well beyond capping enzyme TPases (Gong et al, 2006; Jain and Shuman, 2008). Amino acid sequence comparisons and mutational analyses of metal-dependent RNA triphosphatases of fungi, microsporidia, protozoa and Chlorella virus highlighted conservation of the β strands and essential amino acids that compose the active site tunnel of yeast Cet1, implying a common structure and evolutionary origin for these enzymes (Shuman, 2002), a view later fortified by the structure of mimivirus TPase (Benarroch et al, 2008).…”

Structure of bacterial LigD 3′-phosphoesterase unveils a DNA repair superfamily

“…The five top DALI "hits," with relatively feeble Z scores of 3.6 to 4.2, were members of the triphosphate tunnel metalloenzyme (TTM) superfamily of phosphohydrolases, which are composed of an 8-stranded antiparallel β barrel of quite different folding topology (15,16). The TTM barrel has a wider aperture and an extremely hydrophilic interior that binds the divalent cation and polyphosphorylated substrate on which the TTM enzymes act (17).…”