The Crystal Structure of the Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae Reveals the Basis for Substrate Specificity

Ugochukwu, E.; Lovering, Andrew L.; Mather, O.C.; Young, Thomas W.; White, Scott A.

doi:10.1016/j.jmb.2007.05.066

Cited by 39 publications

(39 citation statements)

References 57 publications

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“…Yeast Ppx1 and E. coli PPX are metal-dependent exopolyphosphatases that cleave the terminal phosphoanhydride linkage to yield P i as the sole product of each catalytic cycle (31,32). The structures of yeast Ppx1 (33) and E. coli PPX (34) have been solved; they are unrelated to each other and bear no resemblance to the TTM fold. Yeast Ppn1 is an endopolyphosphatase that cleaves long polyphosphate chains internally to yield shorter polyphosphates, ultimately forming a mixture of P i and PPP i as the end products (35).…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Jain

Shuman

2008

Journal of Biological Chemistry

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“…4B). A structural similarity search using the DALI server (33) revealed that the globular subdomain is similar to that of the ssDNA 5Ј-3Ј exonuclease RecJ (34) and exopolyphosphatase (35), with high Z scores of 11.5 and 9.1, respectively. Discussion tRNA Interactions.…”

Section: Position Of the Editing Domain The Editing Domain Of A Fulmentioning

confidence: 99%

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Naganuma

Sekine

Fukunaga

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNA Ala by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AlaRS: AlaRS-⌬C, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helixloop-helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding.crystal structure ͉ dimerization domain ͉ aminoacyl-tRNA synthetase ͉ proofreading ͉ wobble base pair

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“…An exopolyphosphatase activity has also been detected in acidocalcisomes of T. cruzi (16). The crystal structures of bacterial (21)(22)(23) and yeast (24) exopolyphosphatases have been reported recently. In contrast to the bacterial exopolyphosphatases that belong to the sugar kinase/actin/hsp-70 superfamily, the yeast and protozoal exopolyphosphatases belong to the DHH (aspartate, histidine, and histidine) superfamily phosphoesterases to which the family II pyrophosphatases belong.…”

mentioning

confidence: 96%

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

Fang

Ruiz²,

Docampo

et al. 2007

Journal of Biological Chemistry

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We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn 2؉ , high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in longchain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation. Polyphosphate (poly P)8 is a ubiquitous polymer of a few to several hundred orthophosphate residues linked by high-energy phosphoanhydride bonds. Poly P is found in the environment and in all life forms, where it is involved in a number of functions (1, 2). Poly P is essential for bacterial responses to stresses and starvation, as well as for survival and virulence (1, 2). Similar functions in adaptation to stress have been attributed to poly P in eukaryotic cells such as yeast (3, 4), fungi (5), and algae (6 -8). Poly P is also involved in blood clotting (9), eukaryotic cell proliferation (10, 11), and induction of apoptosis in plasma and myeloma cells (12).In Trypanosoma cruzi, the etiologic agent of Chagas disease, most poly P is accumulated in acidocalcisomes. Acidocalcisomes are acidic, calcium-containing organelles that have been demonstrated to be involved in osmoregulation (13-16). Rapid hydrolysis of acidocalcisome poly P occurs when epimastigotes of T. cruzi are exposed to hyposmotic stress (16) resulting in increased osmolarity and swelling of the organelle (15). In addition, a microtubule and cAMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of an aquaporin results in water movement and a regulatory volume decrease (15), indicating a link between acidocalcisomes and osmotic homeostasis.In eukaryotic cells the hydrolysis of poly P is performed by the action of endopolyphosphatases and exopolyphosphatases (1, 2). Genes encoding for exopolyphosphatases from Saccharomyces cerevisiae (17), Trypanosoma brucei (18), and Leishmania major (19) have been cloned and expressed in Escherichia coli, whereas a gene encoding an endopolyphosphatase from S. cerevisiae (20) is the only one that has been cloned and expressed. The L. major exopolyphosphatase (LmPPx) has been characterized recently and demonstrated to be located in acidocalcisomes and in the cytosol (19). An exopolyphosphatase activity has also been detected in acidocalcisomes of T. cruzi (16). The crystal structures...

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The Crystal Structure of the Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae Reveals the Basis for Substrate Specificity

Cited by 39 publications

References 57 publications

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

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