Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
1998-11-16
2001-03-13
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S069100, C435S183000, C435S041000, C530S350000, C536S023100, C536S023200, C536S023500
Reexamination Certificate
active
06200795
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated polypeptides having uroporphyrinogen decarboxylase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
Heme, a chelate complex of protoporphyrin IX and iron, serves as a prosthetic group of hemoproteins. Protoporphyrin IX consists of a porphyrin ring, substituted with four methyl groups, two vinyl groups, and two propionic acid groups, which acquires an iron atom to form heme. The biosynthesis of heme from glycine and succinyl-CoA involves eight enzymatic steps. The fifth enzyme in the biosynthetic pathway is uroporphyrinogen decarboxylase which catalyzes four step-wise decarboxylations of uroporphyrinogen III to form coproporphyrinogen III.
The conversion of an apoprotein into a hemoprotein depends on the availability of heme provided by the heme biosynthetic pathway. The apoprotein form of the hemoprotein combines with heme to produce the active hemoprotein. The active hemoprotein acquires a conformation which makes the hemoprotein more stable than the apoprotein to proteolytic attack. If the amount of heme produced by a microorganism is less relative to the amount of the apoprotein produced, the apoprotein will accumulate and undergo proteolytic degradation lowering the yield of the active hemoprotein.
In order to overcome this problem, Jensen showed that the addition of heme or a heme-containing material to a fermentation medium led to a significant increase in the yield of a peroxidase produced by
Aspergillus oryzae
(WO 93/19195). While heme supplementation of a fermentation medium results in a significant improvement in the yield of a hemoprotein, it is non-kosher, costly, and difficult to implement on a large scale.
The overexpression of a gene in the heme biosynthetic pathway of a cell provides an alternative approach for overcoming this problem.
Uroporphyrinogen decarboxylase (also called uro D or uroporphyrinogen III decarboxylase) catalyzes the decarboxylation of all four acetic acid side chains of uroporphyrinogen III to methyl groups to yield coproporphyrinogen III. Uroporphyrinogen decarboxylase genes have been cloned from
Saccharomyces cerevisiae
(Garey et al., 1992,
European Journal of Biochemistry
205:1011-1016; Diflumeri et al., 1993,
Yeast
9: 613-623);
Bacillus subtilis
(Hansson and Hederstedt, 1992,
Journal of Bacteriology
174: 8081-8093);
Escherichia coli
(Nishimura et al., 1993,
Gene
133: 109-113); Synechococcus sp. (Kiel et al., 1992,
DNA Seq.
2: 415-418);
Rhodobacter capsulatus
(Ineichen and Biel, 1995,
Plant Physiology
108: 423); human (Romeo et al., 1986,
Journal of Biological Chemistry
261:9825-9831); and mouse (Wu et al., 1996,
Mammalian Genome
7: 349-352).
It is an object of the present invention to provide improved polypeptides having uroporphyrinogen decarboxylase activity and genes encoding same.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having uroporphyrinogen decarboxylase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 65% identity with the amino acid sequence of SEQ ID NO:2;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or (ii) its complementary strand; or a subsequence thereof of at least 100 nucleotides;
(c) an allelic variant of (a) or (b); and
(d) a fragment of (a), (b) or (c), wherein the fragment has uroporphyrinogen decarboxylase activity.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
REFERENCES:
patent: WO 93/19195 (1993-09-01), None
Gary et al., 1992, European Joournal of Biochemistry 205: 1011-1016.
Diflumeri et al., 1993, Yeast 9: 613-623.
Hansson & Hederstedt, 1992, Journal of Bacteriology 174: 8081-8093.
Nishimura et al., 1993, Gene 133 :109-113.
Kiel et al., 1992, DNA Seq. 2: 415-418.
Ineichen & Biel, 1995, Plant Physiology 108: 423.
Romeo et al., 1986, Journal of Biological Chem. 261: 9825-9831.
Wu et al., 1996, Mammalian Genome 7: 349-352.
Smith & Waterman, 1981, Nucleic Acids Research 10: 197-206.
Garey et al. (1992) Eur. J. Biochem. 205, 1011-1016, May 1992.
Romeo et al. (1986) J. Biol. Chem. 261 (21), 9825-9831, Jul. 25, 1986.
Wu et al. (1996) Mammal. Genome. 7, 349-352, May 1996.
Romana et al. (1987) Nuc. Acid. Res. 15 (17), 7211-7212, Sep. 11, 1987.
Hutson Richard
Lambiris Esq. Elias
Novo Nordisk Biotech Inc.
Prouty Rebecca E.
Stames Robert L.
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