Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-11-02
2003-10-21
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S320100, C435S252300, C435S254110, C435S419000, C435S325000, C536S023100, C536S023200, C536S023600
Reexamination Certificate
active
06635459
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to isolated dirigent proteins and pinoresinol/lariciresinol reductases from
Forsythia intermedia, Tsuga heterophylla
and
Thuja plicata
, to nucleic acid sequences which code for dirigent proteins and pinoresinol/lariciresinol reductases from
Forsythia intermedia, Tsuga heterophylla
and
Thuja plicata
, and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant pinoresinol/lariciresinol reductases, recombinant dirigent protein and their mutants.
BACKGROUND OF THE INVENTION
Lignans are a large, structurally diverse, class of vascular plant metabolites having a wide range of physiological functions and pharmacologically important properties (Ayres, D. C., and Loike, J. D. in
Chemistry and Pharmacology of Natural Products
. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990); Lewis et al., in Chemistry of the Amazon, Biodiversity Natural Products, and Environmental Issues, 588, (P. R. Seidl, O. R. Gottlieb and M. A. C. Kaplan) 135-167, ACS Symposium Series, Washington D.C. (1995)). Because of their pronounced antibiotic properties (Markkanen, T. et al.,
Drugs Exptl. Clin. Res
. 7:711-718 (1981)), antioxidant properties (Fauré, M. et al.,
Phytochemistry
29:3773-3775 (1990); Osawa, T. et al.,
Agric. Biol. Chem
. 49:3351-3352 (1985)) and antifeedant properties (Harmatha, J., and Nawrot, J.,
Biochem. Syst. Ecol
. 12:95-98 (1984)), a major role of lignans in vascular plants is to help confer resistance against various opportunistic biological pathogens and predators. Lignans have also been proposed as cytokinins (Binns, A. N. et al.,
Proc. Natl. Acad. Sci. USA
84:980-984 (1987)) and as intermediates in lignification (Rahman, M. M. A. et al.,
Phytochemistry
29:1861-1866 (1990)), suggesting a critical role in plant growth and development. It is widely held that elaboration of biochemical pathways to lignins/lignans and related substances from phenylalanine (tyrosine) was essential for the successful transition of aquatic plants to their vascular dry-land counterparts (Lewis, N. G., and Davin, L. B., in
Isoprenoids and Other Natural Products. Evolution and Function
, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, D.C. (1994)), some four hundred and eighty million years ago (Graham, L. E.,
Origin of Land Plants
, John Wiley & Sons, Inc., New York, N.Y. (1993)).
Based on existing chemotaxonomic data, lignans are present in “primitive” plants, such as the fern
Blechnum orientate
(Wada, H. et al.,
Chem. Pharm. Bull
. 40:2099-2101 (1992)) and the hornworts, e.g.,
Dendroceros japonicus
and
Megaceros flagellaris
(Takeda, R. et al., in
Bryophytes. Their Chemistry and Chemical Taxonomy
, Vol. 29 (Zinsmeister, H. D. and Mues, R. eds) pp. 201-207, Oxford University Press: New York, N.Y. (1990); Takeda, R. et al.,
Tetrahedron Lett
. 31:4159-4162 (1990)), with the latter recently being classified as originating in the Silurian period (Graham, L. E.,
J. Plant Res
. 109: 241-252 (1996)). Interestingly, evolution of both gymnosperms and angiosperms was accompanied by major changes in the structural complexity and oxidative modifications of the lignans (Lewis, N. G., and Davin, L. B., in
Isoprenoids and Other Natural Products. Evolution and Function
, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, D.C. (1994); Gottlieb, O. R., and Yoshida, M., in
Natural Products of Woody Plants. Chemicals Extraneous to the Lignocellulosic Cell Wall
(Rowe, J. W. and Kirk, C. H. eds) pp. 439-511, Springer Verlag: Berlin (1989)). Indeed, in some species, such as Western Red Cedar (
Thuja plicata
), lignans can contribute extensively to heartwood formation/generation by enhancing the resulting heartwood color, quality, fragrance and durability.
In addition to their functions in plants, lignans also have important pharmacological roles. For example, podophyllotoxin, as its etoposide and teniposide derivatives, is an example of a plant compound that has been successfully employed as an anticancer agent (Ayres, D. C., and Loike, J. D. in
Chemistry and Pharmacology of Natural Products
. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990)). Antiviral properties have also been reported for selected lignans. For example, (−)-arctigenin (Schröder, H. C. et al.,
Z. Naturforsch
. 45c, 1215-1221 (1990)), (−)-trachelogenin (Schröder, H. C. et al.,
Z. Naturforsch
. 45c, 1215-1221 (1990)) and nordihydroguaiaretic acid (Gnabre, J. N. et al.,
Proc. Natl. Acad. Sci. USA
92:11239-11243 (1995)) are each effective against HIV due to their pronounced reverse transcriptase inhibitory activities. Some lignans, e.g., matairesinol (Nikaido, T. et al.,
Chem. Pharm. Bull
. 29:3586-3592 (1981)), inhibit cAMP-phosphodiesterase, whereas others enhance cardiovascular activity, e.g., syringaresinol &bgr;-D-glucoside (Nishibe, S. et al.,
Chem. Pharm. Bull
. 38:1763-1765 (1990)). There is also a high correlation between the presence, in the diet, of the “mammalian” lignans or “phytoestrogens”, enterolactone and enterodiol, formed following digestion of high fiber diets, and reduced incidence rates of breast and prostate cancers (so-called chemoprevention) (Axelson, M., and Setchell, K. D. R.,
FEBS Lett
. 123:337-342 (1981); Adlercreutz et al.,
J. Steroid Biochem. Molec. Biol
. 41:3-8 (1992); Adlercreutz et al.,
J. Steroid Biochem. Molec. Biol
. 52:97-103 (1995)). The “mammalian lignans,” in turn, are considered to be derived from lignans such as matairesinol and secoisolariciresinol (Boriello et al.,
J. Applied Bacteriol
., 58:3743 (1985)).
The biosynthetic pathways to the lignans are only now being defined, although there are no prior art reports of the isolation of enzymes or genes involved in the lignan biosynthetic pathway. Based on radiolabeling experiments with crude enzyme extracts from
Forsythia intermedia
, it was first established that entry into the 8,8′-linked lignans, which represent the most prevalent dilignol linkage known (Davin, L. B., and Lewis, N. G., in
Rec. Adv. Phytochemistry
, Vol. 26 (Stafford, H. A., and Ibrahim, R. K., eds), pp. 325-375, Plenum Press, New York, N.Y. (1992)), occurs via stereoselective coupling of two achiral coniferyl alcohol molecules, in the form of oxygenated free radicals, to afford the furofuran lignan (+)-pinoresinol (Davin, L. B., Bedgar, D. L., Katayama, T., and Lewis, N. G.,
Phytochemistry
31:3869-3874 (1992); Paré, P. W. et al.,
Tetrahedron Lett
. 35:47314734 (1994)) (FIG.
1
).
Bimolecular phenoxy radical coupling reactions, such as the stereoselective coupling of two achiral coniferyl alcohol molecules to afford the furofuran lignan (+)-pinoresinol, are involved in numerous biological processes. These are presumed to include lignin formation in vascular plants (M. Nose et al.,
Phytochemistry
39:71 (1995)), lignan formation in vascular plants (N. G. Lewis and L. B. Davin,
ACS Symp. Ser
. 562:202 (1994); P. W. Paré et al.,
Tetrahedron Lett
. 35:4731 (1994)), suberin formation in vascular plants (M. A. Bernards et al.,
J. Biol. Chem
. 270:7382 (1995)), fruiting body development in fungi (J. D. Bu'Lock et al.,
J. Chem. Soc
. 2085 (1962)), insect cuticle melanization and sclerotization (M. Miessner et al.,
Helv. Chim. Acta
74:1205 (1991); V. J. Marmaras et al.,
Arch. Insect Biochem. Physiol
. 31:119 (1996)), the formation of aphid pigments (D. W. Cameron and Lord Todd, in
Organic Substances of Natural Origin. Oxidative Coupling of Phenols
, W. I. Taylor and A. R. Battersby, Eds. (Dekker, New York, 1967), Vol. 1, p.203), and the formation of algal cell wall polymers (M. A. Ragan,
Phytochemistry
23:2029 (1984)).
In contrast to the marked regiochemical and/or stereochemical specificities observed in the biosynthesis of the foregoing lignin and lignan substances in vivo, all previously described chemical (J. Iqbal et al.,
Chem. Rev
. 94:519 (1994)) and enzymatic (K. Freude
Davin Laurence B.
Dinkova-Kostova Albena T.
Ford Joshua D.
Fujita Masayuki
Gang David R.
Achutamurthy Ponnathapu
Christensen O'Connor Johnson & Kindness PLLC
Kerr Kathleen M
Washington State University Research Foundation
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