Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1997-03-12
1999-01-19
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4353201, 4352523, 43525411, 435325, 536 232, C12N 916, C12N 1500, C12N 120, C07H 2104
Patent
active
058612963
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an extremely thermostable enzyme. More specifically, it relates to a thermostable inorganic pyrophosphatase obtainable from Thermococcus litoralis, to the recombinant DNA which encodes this enzyme and to the production of this enzyme from the recombinant DNA.
BACKGROUND OF THE INVENTION
Inorganic pyrophosphatase (pyrophosphate phosphohydrolase) (PPase) is an enzyme that plays an important role in energy metabolism. This enzyme is responsible for the hydrolysis of pyrophosphate (PP.sub.i) which is formed principally as the product of the many biosynthetic reactions that utilize nucleoside triphosphates, such as DNA and RNA synthesis, coenzyme synthesis and the activation of amino acids and fatty acids. This enzyme is considered to maintain the forward direction of these reactions and is vital for maintaining viability. For a review of microbial inorganic pyrophosphatases, see, Lahti, R., Microbiol. Review (1983) 47:169-179, the disclosure of which is hereby incorporated by reference herein.
Inorganic pyrophosphatases occur widely in nature. The two best-studied PPases are those from Saccharomyces cerevisiae and Escherichia coli. See, for example, Cooperman, B. S., Methods Enzymol. (1982) 87:526-548, and Josse, J. and Wong, D. C. K. in Enzymes (1971), 3rd Ed., Vol. 4 (Boyer, P. D., ed.) pp. 499-527, Academic Press, New York. Research (the disclosure of which is hereby incorporated by reference herein), has also been conducted on the isolation and purification of inorganic pyrophosphatases from other microorganisms. Four PPases have been isolated from the Archaea: Methanobacteriun thermoautotrophicum (strain .DELTA.H) in Alebeek, et al., Biochim. Biophys. Acta (1994) 1206:231-239, the disclosure of which is hereby incorporated by reference herein; Methanothrix soehngenii in Jetten, et al., Arch. Microbiol. (1992) 157:284-289, the disclosure of which is hereby incorporated by reference herein; Thermoplasma acidophilum in Richter and Schafer, Eur. J. Biochem. (1992) 209:343-349, the disclosure of which is hereby incorporated by reference herein; and Sulfolbus acidocaldarius strain 7 in Wakagi, et al., Biochim. Biophys. Acta (1992) 1120:289-296, the disclosure of which is hereby incorporated by reference herein, and S. acidocaldarius (DSM 639) in Meyer, et al., Arch. Biochem. Biophys. (1995) 319:149-156, the disclosure of which is hereby incorporated by reference herein. Furthermore, the 3-dimensional structure of three PPases has been determined by X-ray crystallography: Saccharomyces cerevisiae (Baker's yeast) in Arutiunian, et al., Dokl. Akad. Nauk. SSSR (1981) 258:1481, the disclosure of which is hereby incorporated by reference herein; E. coli in Kankare, et al., Protein Engineering (1994) 7:823-830, the disclosure of which is hereby incorporated by reference herein; and Thermus thermophilus in Teplyokov, et al., Protein Science (1994) 3:1098-1107, the disclosure of which is hereby incorporated by reference herein.
Two different categories of inorganic pyrophosphatases have been differentiated: soluble (cytoplasmic) and membrane-bound. This differentiation is based not only on cellular localization, but on subunit structure. Cytoplasmic PPases are generally oligomeric proteins consisting of identical subunits. Enzymes from eubacterial and archaeal sources tend to be 19 to 23 kDa subunits and exist as either a tetramer or hexamer. See Alebeek, et al., supra (the disclosure of which is hereby incorporated by reference herein). Dissociation to dimers and trimers has been shown to exist in the absence of divalent cations, Icheba et al., J. Biochem. (1990) 108: 572-578. Eucaryotic enzymes are homodimers of 32 to 35 kDa and are exclusively dimers. The only known exception to these rules is the PPase from Methanothrix soehngenii that posses an .alpha..sub.2 .beta..sub.2 subunit structure, Jetten, et al., supra (the disclosure of which is hereby incorporated by reference herein).
All known PPases require the presence of divalent metal cations, with magnesi
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Lennox Tricia
Sears Lauren E.
Slatko Barton E.
Hobbs Lisa J.
New England Biolabs Inc.
Wax Robert A.
Williams Gregory D.
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