Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-07-21
2003-02-11
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S281000, C800S312000, C800S314000, C800S320100, C800S320300, C435S419000, C536S023200, C536S023600
Reexamination Certificate
active
06518488
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of plant biochemistry, particularly as it pertains to the &bgr;-oxidation pathway. More specifically, the invention relates to nucleic acid molecules that encode proteins and fragments of proteins associated with the &bgr;-oxidation pathway, the proteins and fragments of proteins so encoded, and antibodies capable of binding the proteins. The invention also relates to methods of using the nucleic acid molecules, proteins, and fragments of proteins.
BACKGROUND OF THE INVENTION
The degradation of fatty acids occurs by the &bgr;-oxidation pathway &bgr;-oxidation plays an important role in the metabolism of stored seed lipids during seed germination and early seedling growth (Cooper and Beevers,
J. Biol. Chem.,
244:3514-3520 (1969)). The end-products of lipid breakdown provide energy to the growing seedling until it becomes photosynthetic. &bgr;-oxidation is not, however, restricted to the seedling growth stage of plant development. This process occurs in several different tissues and its possible physiological roles include energy generation, turnover of membrane lipids and the removal of toxic fatty acids (Gerhardt,
Physiol. Veg.,
24:397-410 (1986); Tramantano et al.,
Phytochemistry
36:19-21 (1994)). It also plays a role in membrane turnover during senescence (Wanner et al.,
Plant Sci.,
78:199-206 (1991)). Therefore, &bgr;-oxidation is a consistent basic function of all cells of a plant (Gerhardt,
Planta
159:238-246 (1983)).
Fatty acid oxidation is reported in three systems; mitochondrial, peroxisomal and bacterial. Mitochondrial and peroxisomal &bgr;-oxidation occurs in animal cells, peroxisomal &bgr;-oxidation occurs in plant cells and bacterial &bgr;-oxidation is reported to differ from eukaryotic &bgr;-oxidation. Peroxisomal &bgr;-oxidation is similar to the mitochondrial &bgr;-oxidation, except that carnitine has not been reported to be required. In mitochondria, long chain fatty acids are activated by acyl-CoA synthetase on the mitochondrial outer membrane and acyl groups of the CoA esters are transported into the matrix by carnitine acyltransferase. Mitochondrial &bgr;-oxidation has been reported as cyclic repetition of four basic reactions catalyzed by a long, medium and short chain acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl CoA dehydrogenase and 3-ketoacyl-CoA thiolase. The reported substrates of &bgr;-oxidation enzymes are coenzyme A (CoA) derivatives of fatty acid. In peroxisomes, fatty acids have been reported to be activated by acyl-CoA synthetase (Shindo and Hashimoto,
J. Biochem.
84:1177-1181 (1978); Krisans et al.,
J. Biol. Chem.
255:9599-9607 (1980). Acyl-CoA esters have been reported to be degraded by &bgr;-oxidation cycle. &bgr;-oxidation has been reported to be catalyzed by acyl-CoA oxidase, enoyl CoA isomerase/enoyl-CoA hydratase/3-hydroxylacyl-CoA dehydrogenase.
Acyl-CoA oxidase (EC 1.3.3.6) is the first reported enzyme of the fatty acid &bgr;-oxidation pathway. This enzyme catalyzes the desaturation of acyl-CoAs longer than eight carbons to 2-trans-enoyl-CoAs, by donating electrons directly to molecular oxygen and releasing H
2
O
2
(Lazarow et al., 1976). Acyl-CoA oxidase substrate has been reported as acyl moieties of more than eight carbon atoms (Osumi et al.,
J. Biochem.
87:1735-1746 (1980).
Bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase is the second reported enzyme of the peroxisomal &bgr;-oxidation pathway. Enoyl-CoA hydratase catalyzes hydration of double bond to form 3-L-hydroxyacyl-CoA. 3-hydroxyacyl-CoA dehydrogenase catalyzes NAD
+
dependent dehydrogenation of &bgr;-hydroxy-acyl-CoA resulting in the formation of the corresponding &bgr;-ketoacyl-CoA. Originally, bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase was reported in rat liver as a monomeric protein with two enzyme activities (Osumi and Hashimoto,
Biochem. Biophys. Res. Commun.
89:580-584 (1979). Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase has also been reported as a trifunctional protein with an enoyl-CoA isomerase activity in addition to hydratase and dehydrogenase activity (Palorassi and Hiltunen,
J. Biol. Chem.
265:2446-2449 (1990). Enoyl CoA isomerase/enoyl-CoA hydratase/3-hydroxylacyl-CoA dehydrogenase has also been reported in bovine liver, pig heart and human liver (Fong and Schulz,
Methods Enzymol.
71:390-398 (1981); Furuta et al.,
J. Biochem.
88:1059-1070 (1980); Reddy et al.,
Proc. Natl. Acad. Sci.
(
USA
) 84:3214-3218 (1987); Osumi and Hashimoto,
J. Biol. Chem.
262: 8138-8143 (1979). Rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA isomerase has been reported to contain seven exons. Exons one through five, are reported at the amino terminal to constitute a hydratase domain. 3-hydroxyacyl CoA dehydrogenase activity is reported in exons six and seven. 3-hydroxyacyl CoA dehydrogenase activity has been reported to be present in a 722 amino acid polypeptide (Ishii et al,
J. Biol. Chem.
262:8144-8150 (1987); Osumi et al.,
J. Biol. Chem.
260:8905-8910 (1985).
3-Ketoacyl-CoA thiolase is reported to catalyze the last step of fatty acid &bgr;-oxidation, resulting in C&agr;-C&bgr; cleavage yielding acetyl-CoA and new acyl-CoA with two fewer carbons the original one. Two types of mitochondrial thiolases have been reported which differ chain length specificity: 3-ketoacyl CoA thiolase (also known as thiolase I) and acetoacetyl-CoA thiolase (EC 2.3.1.9) (also known as thiolase II). 3-Ketoacyl-CoA-thiolase (EC 2.3.1.16) has reported activity on substrates ranging from acetoacetyl-CoA to long-chain 3-ketoacyl-CoAs at low concentration (Middleton,
Methods Enzymol.
35:128-136 (1975; Staack et al,
J. Biol. Chem.
253:1827-1931 (1978). Thiolase has been reported as a tetramer. Rat mitochondrial 3-ketoacyl-CoA thiolase has been reported to have a molecular weight of 41866 Kd (Arakawa et al,
EMBO J.
6:1361-1366 (1987). Peroxisomal 3-ketoacyl-CoA thiolase has been reported in rat liver as a homodimer with a molecular mass of 89 kDa.
Mitochondrial 3-ketoacyl-CoA thiolases and mitochondrial and cytosolic acetoacetyl-CoA specific thiolases have been reported as homotetramers, each subunit is about 40 kDa (Miyazawa et al.,
Eur. J. Biochem.
103:589-596 (1980). Genes encoding these enzymes have been reported (Hijikata et al.,
J. Biol. Chem.
262:8151-8158 (1990). A rat peroxisomal 3-ketoacyl-CoA thiolase and a mitochondrial 3-ketoacyl-CoA thiolase have been reported which contain cysteine residues that are important for substrate binding (Hijikata et al.,
J. Biol. Chem.
262:8151-8158 (1987); Arakawa et al.,
EMBO J.
6:1361-1366 (1987)). Thiolases from different species have been reported to have an essential sulfhydryl serving as an acyl acceptor during the thiolytic cleavage (Gilbert et al.,
J. Biol. Chem.
256:7371-7377 (1981).
The isolation and identification of cDNAs encoding proteins in the &bgr;-oxidation pathway will help to confirm the activities of the enzymes encoded and their substrate specificities. Expression studies may be used, for example, to determine fatty acid substrate chain length specificity. There are multiple isozymes of acyl-CoA oxidase and these isozymes show specificity towards short, medium and long chain fatty acyl-CoAs (Hooks et al.,
Biochem J.,
320:607-614 (1996); Hooks et al.,
Plant J.,
20:1-13 (1999)). It is likely that long chain specificity may be required for &bgr;-oxidation of seed lipids and broad specificity may be required for other stages.
The present invention provides complete and partial cDNAs encoding &bgr;-oxidation pathway enzymes. The invention also provides protein and fragment molecules with amino acid sequences in the &bgr;-oxidation pathway. The nucleic acid molecules, drawn from soy and maize, may be used to understand the different functions of b-oxidation during plant growth and development, leading to the development of nutritionally and agriculturally enhanced crops and products.
SUMMARY OF THE INVENTION
The present invention includes and
Agarwal Ameeta
Lahiri Devlina
Liu Jingdong
Arnold & Porter
Lavin, Jr. Lawrence M.
McElwain Elizabeth F.
Monsanto Technology LLC
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