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Modeling Studies of Potato Nucleoside Triphosphate Diphosphohydro- Lase Ntpdase1: an Insight Into the Catalytic Mechanism

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Modeling Studies of Potato Nucleoside Triphosphate Diphosphohydro- Lase Ntpdase1: an Insight Into the Catalytic Mechanism Vol. 55 No. 1/2008, 141–150 on-line at: www.actabp.pl Regular paper Modeling studies of potato nucleoside triphosphate diphosphohydro- lase NTPDase1: an insight into the catalytic mechanism Anna Kozakiewicz1, Piotr Neumann1, Mariusz Banach2, Michał Komoszyński2 and Andrzej Wojtczak1 1Department of Crystallochemistry and Biocrystallography, Institute of Chemistry, Toruń, Poland; 2Department of Biochemistry, Institute of Biology, N. Copernicus University, Toruń, Poland Received: 13 November, 2007; revised: 21 January, 2008; accepted: 05 March, 2008 available on-line: 11 March, 2008 Nucleoside triphosphate diphosphohydrolase — NTPDase1 (apyrase, EC 3.6.1.5) was modeled based on sequence homology. The single polypeptide chain of apyrase is folded into two do- mains. The putative catalytic site with the apyrase conserved regions (ACR 1–5) is located be- tween these two domains. Modeling confirmed that apyrase belongs to the actin superfamily of proteins. The amino acids interacting with the nucleoside triphosphate substrate and probably involved in the catalyzed hydrolysis were identified. The proposed two-step catalytic mechanism of hydrolysis involves Thr127 and Thr55 as potential nucleophilic factors responsible for the cleavage of the Pγ and Pβ anhydride bonds, respectively. Their action seems to be assisted by Glu170 and Glu78 residues, respectively. The presence of two nucleophiles in the active site of apyrase explains the differences in the hydrolytic activity between apyrases and other enzymes belonging to the NTPDase family. Keywords: NTPDase1, potato apyrase, homology modeling, enzymatic mechanism INTRODUCTION ological importance of mechanisms controling nu- cleotide concentration. All NTPDases belong to the At least three groups of enzymes regulate the sub-class of ATP diphosphohydrolases (EC 3.6.1). levels of cellular and extracellular nucleotides and These enzymes might be either soluble (Oses et al., nucleosides: nucleoside triphosphate diphosphohy- 2004) or bound to the membranes. The catalytic site drolases (NTPDases), pyrophosphatases/phosphodi- of enzymes occurring in the plasma membranes or esterases (NPPases), and alkaline phosphatases. The in membranes of subcellular structures is located at first group hydrolyzes only compounds with py- the extracytoplasmic side of the membranes (Hanada rophosphate bonds. Enzymes belonging to the two & Guidotti, 1996; Robson et al., 2006). In literature latter groups hydrolyze anhydride or ester bonds apyrases are classified as NTPDases1 based on their of nucleotides. Therefore, depending on the group similar velocity of the ATP and ADP hydrolysis. of enzymes, the products of the catalyzed reaction NTPDases are a structurally and catalytically might be either orthophosphate or pyrophosphate homogeneous group of enzymes. Genes encoding ions and the corresponding nucleoside monophos- these proteins have been found in vertebrates, in- phate (NMP), nucleoside diphosphate (NDP) or vertebrates, plants and protozoans. The sequence nucleoside (Robson et al., 2006). High diversity of identity among mammalian NTPDases varies be- enzymes degrading nucleotides indicates the physi- tween 24 and 96%, while the identity between apy- Corresponding author: Andrzej Wojtczak, Department of Crystallochemistry and Biocrystallography, Institute of Chem- istry, N. Copernicus University, Gagarina 7, 87-100 Toruń, Poland; fax: (48) 56 654 2477; e-mail: [email protected] Abbreviations: ACR, apyrase conserved region; ATPase, adenosine triphosphate hydrolase; ENTPDase, ecto-nucleoside triphosphate diphosphohydrolase; NDP, nucleoside diphosphate; NMP, nucleoside monophosphate; NTP, nucleoside tri- phosphate; NTPDase, nucleoside triphosphate diphosphohydrolase; NPPase, pyrophosphatase/phosphodiesterase; PDB, Protein Data Base; Pi, inorganic phosphate; PPX/GPPA, exopolyphosphatase/guanosine pentaphosphate phosphohydro- lase; rms, root mean square; SA MD, simulated annealing molecular dynamics. 142 A. Kozakiewicz and others 2008 rases (ATP diphosphohydrolase — EC 3.6.1.5) from 1999). Therefore, the enzymes belonging to that su- pea or potato and mammalian NTPDases is only perfamily might be structural templates for building 23–24% (Lavoie et al., 2004). These two groups of an apyrase model. Lately, a theoretical model of hu- enzymes seem to have evolved from a common an- man NTPDase3 was obtained based on the exopoly- cestor, but have separated at an early stage of evolu- phosphatase 1T6C template (Ivanenkov et al., 2005). tion (Lavoie et al., 2004). Five characteristic apyrase The aim of the reported research was to construct conserved regions (ACR) are found in the sequences a 3-dimensional model of apyrase based on the se- of NTPDases (Smith & Kirley, 1999). Mutagenesis quence homology to members of the actin super- data show that these motifs are involved in sub- family, and to propose a reaction mechanism based strate recognition, binding and hydrolysis (Kirley et on the location of residues potentially involved in al., 2006). NTPDases reveal a low substrate specifi- catalysis. city, hydrolyzing purine and pyrimidine nucleoside di- and triphosphates. This might be explained by their high similarity in the ACR regions. However, METHODS different NTPDases hydrolyze nucleoside di- and triphosphates with different velocity (Kukulski & The potato apyrase sequence derived from Komoszyński, 2003; Kukulski et al., 2005). No con- the corresponding cDNA sequence (Hanada & Gui- sistent relations has been found between the residue dotti, 1996) was used for similarity analysis carried mutations in ACR and the NTP or NDP preferences out via the Fold Recognition Meta-Server http:// in the NTPDase family of enzymes (Robson et al., www.genesilico.pl/meta (Kurowski & Bujnicki, 2006). 2003), which uses freely available services for pro- The catalytic mechanism of ATP diphospho- tein secondary structure prediction and fold recogni- hydrolases is unclear. These enzymes, like potato tion, among them MGENTHREADER (McGuffin & apyrase (NTPDase1), hydrolyze ATP and ADP with Jones, 2003) and PSI-PRED (Jones, 1999). Only one similar velocities. The Ksh value, which is the ratio sequence — PPX/GPPA exopolyphosphatase (Kris- of ATP to ADP hydrolysis velocities, is between 1 tensen et al., 2004) — was chosen as the principal and 0.5 (Komoszyński & Wojtczak, 1996; Kukulski & modeling template due to the highest score given Komoszyński, 2003; Kukulski et al., 2005). There are consistently by all fold recognition services and sig- two general models of ATP hydrolysis. In the first nificant score values allowing homology prediction. model, ATP and ADP are hydrolyzed by the same In particular, the similarity scoring function calcu- domain of apyrase. The second model is based on lated with MGENTHREADER was 0.958 and indi- the assumption that ADP produced in one active site cated a reliable sequence alignment. Kristensen et is redirected to the other site and then hydrolyzed al. (2004) described two structures of that exopoly- to AMP. The first model is supported by results of phosphatase in an open and closed conformation kinetic research on these enzymes. Hydrolysis of (PDB codes: 1T6C and 1T6D, respectively). The rms ATP or ADP by ENTPDase1 results in AMP and 1 difference between those structures is 0.64 Å, as or 2 Pi ions even after very short time. The KM for calculated for 303 Cα positions. Modeling of the 3- ATP and ADP are equal (Kukulski & Komoszyński, dimensional structure of NTPDase1 was performed 2003). The second model was excluded by results of based on both these structures. The PPX/GPPA ex- hydrolysis of an ATP-ADP mixture with the starting opolyphosphatase also belongs to the actin super- ATP concentration approximately 103 times higher family (Kristensen et al., 2004). The sequence align- than that of ADP (Kukulski & Komoszyński, 2003), ments produced by other servers (FFAS, FUGUE) still leading to the immediate production of AMP. (Rychlewski et al., 2000; Shi et al., 2001) were similar Such a mechanism would require a very high en- to that obtained with MGENTHREADER. zyme affinity for ADP and a low affinity for ATP, in Potato apyrase is larger than PPX/GPPA ex- contradiction to the determined KM values. Also, the opolyphosphatase due to the presence of some long high catalytic activity of apyrase (Hanada & Gui- insertions (Met1–Glu42, Glu224–Arg296 regions) that dotti, 1996) and the non-additive hydrolysis of ATP are absent in the template structure. The Met1–Glu42 and ADP in equimolar concentrations (Kukulski et region was modeled using the Arg8–Lys47 N-termi- al., 2005) oppose such a mechanism. So far there is nal fragment of ribosomal protein S15 (Clemons et no report on the catalytic mechanism of apyrase at al., 1998) (PDB code: 1A32). This protein was cho- the molecular level. sen based on similarity of the secondary structure Determination of the tertiary structure of and subsequent sequence-sequence alignment with NTPDases might give an insight into the catalytic T-coffee (Notredame et al., 2000). A large insertion mechanism of these enzymes. It was reported that (Glu224–Arg296) occurs in the position correspond- these nucleotidases belong to the actin/Hsp70/sug- ing to sub-domain 4 of exopolyphosphatase (Fig. 1). ar kinase superfamily of proteins (Smith & Kirley, An analogous domain with a similar length but low Vol. 55 NTPDase1: an insight into the catalytic mechanism 143 a) 1 20 40 ACR1 60 80 b) MLNQNSHFIFIILAIFLVLPLSLLSKNVNAQIPLRRHLLSHESEHYAVIFDAGSTGSRVHVFRFDEKLGLLPIGNNIEYF REIIEQFKVHENDTGSPEVQIAILTEQIN---NLNEHLRVHKK
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