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2013 | 60 | 2 | 249-254
Article title

Hint2, the mitochondrial nucleoside 5'-phosphoramidate hydrolase; properties of the homogeneous protein from sheep (Ovis aries) liver

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Adenosine 5'-phosphoramidate (NH2-pA) is a rare natural nucleotide and its biochemistry and biological functions are poorly recognized. All organisms have proteins that may be involved in the catabolism of NH2-pA. They are members of the HIT protein family and catalyze hydrolytic splitting of NH2-pA to 5'-AMP and ammonia. At least five HIT proteins have been identified in mammals; however, the enzymatic and molecular properties of only Fhit and Hint1 have been comprehensively studied. Our study focuses on the Hint2 protein purified by a simple procedure to homogeneity from sheep liver mitochondrial fraction (OaHint2). Hint1 protein was also prepared from sheep liver (OaHint1) and the molecular and kinetic properties of the two proteins compared. Both function as homodimers and behave as nucleoside 5'-phosphoramidate hydrolases. The molecular mass of the OaHint2 monomer is 16 kDa and that of the OaHint1 monomer 14.9 kDa. Among potential substrates studied, NH2-pA appeared to be the best; the Km and kcat values estimated for this compound are 6.6 μM and 68.3 s-1, and 1.5 μM and 11.0 s-1 per natively functioning dimer of OaHint2 and OaHint1, respectively. Studies of the rates of hydrolysis of different NH2-pA derivatives show that Hint2 is more specific towards compounds with a P-N bond than Hint1. The thermostability of these two proteins is also compared.
Physical description
  • Andrews P (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J 91: 222-233.
  • Banerjee H, Palenchar JB, Łukaszewicz M, Bojarska E, Stępiński J, Jemielity J, Guranowski A, Ng S, Wah DA, Darżynkiewicz E, Bellofatto V (2009) Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins. RNA 15: 1554-1564.
  • Barnes LD, Garrison PN, Siprashvili Z, Guranowski A, Robinson AK, Ingram SW, Croce CM, Ohta M, Huebner K (1996) Fhit, a putative tumor suppressor in humans, is a dinucleoside 5', 5'''-P1,P3-triphosphate hydrolase. Biochemistry 35: 11529-11535.
  • Bieganowski P, Garrison PN, Hodawadekar SC, Faye G, Barnes LD, Brenner C (2002) Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of kin28, Ccl1, and Tfb3. J Biol Chem 277: 10852-10860.
  • Carmi-Levy I, Yannay-Cohen N, Kay G, Razin E, Nechushtan H (2008) Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells. Mol Cell Biol 28: 5777-5784.
  • Chambers RW, Moffatt JG (1958) The synthesis of adenosine-5' and uridine-5'-phosphoramidates. J Am Chem Soc 80: 3752-3755.
  • Cohen LS, Mikhli C, Friedman C, Jankowska-Anyszka M, Stępiński J, Darżynkiewicz E, Davis RE (2004) Nematode m7GpppG and m32,2,7GpppG decapping: activities of Ascaris embryos and characterization of C. elegans scavenger DcpS. RNA Biol 10: 1609-1624.
  • Fankhauser H, Berkowitz GA, Schiff JA (1981a) A nucleotide with the properties of adenosine 5'-phosphoramidate from Chlorella cells. Biochem Biophys Res Commun 101: 524-532.
  • Fankhauser H, Schiff JA, Garber LJ (1981b) Purification and properties of adenylyl sulfate ; ammonia adenylyltransferase from Chlorella catalyzing the formation of adenosine 5'-phosphoramidate from adenosine 5'-phosphosulfate and ammonia. Biochem J 195: 545-560.
  • Guranowski A (1979) Plant adenosine kinase: purification and some properties of the enzyme from Lupinus luteus seeds. Arch Biochem Biophys 196: 220-226.
  • Guranowski A, Wojdyla AM, Pietrowska-Borek M, Bieganowski P, Khurs EN, Cliff MJ, Blackburn GM, Błaziak D, Stec WJ (2008) Fhit proteins can also recognize substrates other than dinucleoside polyphosphates. FEBS Lett 582: 3152-3158.
  • Guranowski A, Wojdyla AM, Zimny J, Wypijewska A, Kowalska J, Jemielity J, Davis RE, Bieganowski P (2010a) Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP). FEBS Lett 584: 93-98.
  • Guranowski A, Wojdyla AM, Zimny J, Wypijewska A, Kowalska J, Łukaszewicz M, Jemielity J, Darżynkiewicz E, Jagiełło A, Bieganowski P (2010b) Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins. New J Chem 34: 888-893.
  • Guranowski A, Wojdyla AM, Rydzik AM, Stępiński J, Jemielity J (2011) Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme. Acta Biochim Pol 58: 131-136.
  • Korsisaari N, Mäkelä TP (2000) Interactions of Cdk7 and Kin28 with Hint/PKC-1 and Hnt1 histidine triad proteins. J Biol Chem 275: 34837-34840.
  • Korsisaari N, Rossi DJ, Luukko K, Huebner K, Henkemeyer M, Mäkelä TP (2003) The histidine triad protein Hint 1 is not required for murine development or Cdk7 function. Mol Cell Biol 23: 3929-3935.
  • Kowalska J, Osowniak A, Zuberek J, Jemielity J (2012) Synthesis of nucleoside phosphosulfates. Bioorg Med Chem Lett 22: 3661-3664.
  • Kuba M, Okizaki T, Ohmori H, Kumon A (1994) Nucleoside monophosphoramidate hydrolase from rat liver: purification and characterization. Int J Biochem 26: 235-245.
  • Lenglet S, Antigny F, Vetterli L, Dufour JF, Rossier MF (2008) Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis. Endocrinology 149: 5461-5469.
  • Linster CL, Van Schaftingen E, Hanson AD (2013) Metabolite damage and its repair or pre-emption. Natue Chem. Biol. 9: 72-78.
  • Lohrmann R (1977) Formation of nucleoside 5'-phosphoramidates under potentially prebiological conditions. J Mol Evol 10: 137-154.
  • Maize KM, Wagner CR, Finzel BC (2013) Structural characterization of human histidine triad nucleotide binding protein 2 (hHint2), a member of the histidine triad (HIT) superfamily. doi: 10.1111/febs.12330.
  • Martin J, St-Pierre MV, Dufour JF (2011) Hit proteins, mitochondria and cancer. Biochim Biophys Acta 1807: 626-632.
  • Martin J, Magnino F, Schmidt K, Piguet AC, Lee JS, Semela D, St-Pierre MV, Ziemiecki A, Cassio D, Brenner Ch, Thorgeirsson SS, Dufour JF (2006) Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellolar carcinoma. Gastroenterology 130: 2179-2188.
  • McLennan AG (2000) Dinucleoside polyphosphates-friend or foe? Pharmacol Ther 87: 73-89.
  • Mejbaum-Katzenellenbogen W (1955) Turbidymetryczna mikrometoda oznaczania białek taniną. Acta Biochim Pol 2: 279-296.
  • Mukaiyama T, Hashimoto M (1971) Phosphorylation by oxidation-reduction condensation. Preparation of active phosphorylating reagents. Bul Chem Soc Japan 44: 2284.
  • Ozga M, Dolot R, Janicka M, Kaczmarek R, Krakowiak A (2010) Histidine triad nucleotide-binding protein 1 (Hint-1) phosphoramidase transforms nucleoside 5'-O-phosphorothioates to nucleoside 5'-phosphates. J Biol Chem 285: 40809-40818.
  • Romanowska J, Sobkowski M, Szymańska-Michalak A, Kołodziej K, Dąbrowska A, Lipniacki A, Piasek A, Pietrusiewicz ZM, Figlerowicz M, Guranowski A, Boryski J, Stawiński J, Kraszewski A (2011) Aryl H-phosphonates 17: (N-Aryl)phosphoramidates of pyrimidine nucleoside analogues and their synthesis, selected properties, and anti-HIV activity. J Med Chem 54: 6482-6591.
  • Rossomando EF, Hadjimichael J (1986) Characterization and cAMP inhibition of a lysyl-(N-ε-5'-phospho) adenosyl phosphoamidase in Dictyostelium discoideum. Int J Biochem 18: 481-484.
  • Weiske J, Huber O (2005) The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF - β-catenin-mediated transcription. J Cell Sci 118: 3117-3129.
  • Weiske J, Huber O (2006) The histidine triad protein Hint1 triggers apoptosis independent of its enzymatic activity. J Biol Chem 281: 27356-27366.
  • Wittman R (1963) Die Reaktion der Phosphorsäuren mit 2,3-Dinitro-fluorobenzol. I: Eine neue Synthese von Monofluorophosphorsäurenmonoestern. Chem Ber 96: 771-779.
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