Aryl propenal double bond reductase

Details Aryl propenal double bond reductase Aryl propenal double bond reductase

2001-03-27

2004-03-09

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S006120, C435S252300, C435S320100, C435S410000, C516S023000, C516S029000, C800S295000

Reexamination Certificate

active

06703229

ABSTRACT:

FIELD OF THE INVENTION
This application relates to aryl propenal double bond reductase proteins, to nucleic acid molecules and vectors encoding aryl propenal double bond reductase proteins, and to methods of altering the composition of plant material, such as wood, by altering the level of expression of one or more aryl propenal double bond reductase proteins within a plant.
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. 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.,
58:37-43 (1985)).
The biosynthetic pathways to the lignans are only now being defined. In this regard, loblolly pine (
Pinus taeda
) is a popular and commercially important softwood species in the United States, and has been used as a model for studies on lignin biosynthesis and monolignol regulation. Moreover, its cell suspension cultures accumulate various 8-5′ linked lignans, namely dehydrodiconiferyl alcohol (DDC) and dihydrodehydrodiconiferyl alcohol (DDDC), as well as the 8-8′ linked lignan, pinoresinol, when placed in a culturing medium containing 8% sucrose and 20 mM KI solution. The present inventors have utilized these cell cultures to characterize enzymes involved in the double bond hydrogenation system of DDC and coniferyl alcohol since the resulting metabolites help determine the color, durability and resistance of the wood, particularly the heartwood.
Thus, in one aspect, the present invention provides a cDNA molecule (SEQ ID NO:1) isolated from loblolly pine (
Pinus taeda
) cells, and the encoded aryl propenal double-bond reductase (SEQ ID NO:2). Aryl propenal double-bond reductase (SEQ ID NO:2) expressed from the isolated cDNA molecule (SEQ ID NO:1) was found to regiospecifically reduce the double-bond of dehydrodiconiferyl aldehyde (DDCAL) to afford dihydrodehydrodiconiferyl aldehyde (DDDCAL) in the presence of [4R]-NADPH. The recombinant aryl propenal double-bond reductase (SEQ ID NO:2) was also capable of reducing coniferyl aldehyde to dihydroconiferyl aldehyde, and
P. taeda
soluble enzyme preparations also catalyzed the reduction of dihydroconiferyl aldehyde to give dihydroconiferyl alcohol, i.e., the aryl propenal double-bond reductase (SEQ ID NO:2) acts on both monomeric and dimeric aryl propenals.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides isolated nucleic acid molecules that each: (a) encode an aryl propenal double bond reductase; and (b) hybridize to a nucleic acid molecule consisting of the complement of the nucleic acid sequence set forth in SEQ ID NO:1 under conditions of 1×SSC at 55° C. for one hour. Some nucleic acid molecules of this aspect of the invention are cDNA molecules. An exemplary nucleic acid molecule of the invention is the cDNA molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:1 that encodes the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2.
In another aspect, the present invention provides isolated nucleic acid molecules that each encode an aryl propenal double bond reductase that is at least 50% identical (such as at least 70% identical, or at least 80% identical, or at least 90% identical) to the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2. The present invention also provides vectors that include a nucleic acid molecule of the invention, and host cells (such as plant cells) that include a vector of the invention. Thus, in one embodiment, the present invention provides vectors that each comprise a nucleic acid molecule that: (a) encodes an aryl propenal double bond reductase; and (b) hybridizes to the complement of SEQ ID NO:1 under conditions of 1×SSC, 55° C. for one hour.
In yet another aspect, the present invention provides isolated aryl propenal double bond reductase proteins that are at least 50% identical (such as at least 70% identical, or at least 80% identical, or at least 90% identical) to the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2.
In a further aspect, the present invention provides methods of enhancing the level of aryl propenal double bond reductase in a plant, the methods each comprising the steps of: (a) introducing into a plant an expression vector comprising a nucleic acid molecule that: (1) encodes an aryl propenal double bond reductase; (2) hybridizes to the complement of SEQ ID NO:1 under conditions of 1×SSC at 55° C. for one hour; and (b) expressing the aryl propenal double bond reductase within the plant.
The present invention also provides methods of inhibiting the expression of aryl propenal double bond reductase in a plant, the methods comprising the steps of: (a) introducing into a plant an expression vector that comprises a nucleic acid molecule that is in anti

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