Materials and methods for the alteration of enzyme and...

Details Materials and methods for the alteration of enzyme and... Materials and methods for the alteration of enzyme and...

1999-06-25

2004-07-20

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S419000, C435S468000, C536S023600, C536S034000, C800S281000

Reexamination Certificate

active

06764851

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), pyruvate decarboxylase (PDC) from Arabidopsis, and aldehyde dehydrogenase (ALDH) from Arabidopsis. The present invention also relates to a recombinant vector comprising (i) a nucleic acid sequence encoding an aforementioned enzyme, (ii) an antisense sequence thereto or (iii) a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme or acetyl CoA, or the capacity to produce acetyl CoA, has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. In addition, the present invention relates to a recombinant vector comprising (i) an antisense sequence to a nucleic acid sequence encoding PDC, the E1
&agr;
subunit of pPDH, the E1
&bgr;
subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or ALDH or (ii) a ribozyme that can cleave an RNA molecule encoding PDC, E1
&agr;
pPDH, E1
&bgr;
pPDH, E2 pPDH, mtPDH or ALDH.
BACKGROUND OF THE INVENTION
ACS and pPDH are two enzymes that are responsible for the generation of acetyl CoA in the plastids, e.g., chloroplasts, of plants. ACS generates acetyl CoA as follows:
acetate+ATP+CoASH→acetyl-CoA+AMP+PP
i
,
wherein ATP represents adenine triphosphate, CoASH represents coenzyme A, acetyl-CoA represents acetyl coenzyme A, AMP represents adenine monophosphate, and PP
i
represents inorganic pyrophosphate, and wherein the acetate includes that which results from the conversion of acetaldehyde and NAD
+
to acetate and NADH, wherein the acetaldehyde, in turn, results from the breakdown of pyruvate, which releases CO
2
. pPDH generates acetyl CoA as follows:
pyruvate+CoASH+NAD
+
→acetyl-CoA+CO
2
+NADH,
wherein NAD
+
represents nicotinamide adenine dinucleotide and NADH represents the reduced form of NAD
+
and wherein the pyruvate results from glycolysis. Glycolysis involves the conversion of sugar phosphates, which have been produced from starch, photosynthesis or the importation of triose and hexose phosphates from the cytosol, to pyruvate.
Various studies of relative activity of enzymes in embryos and leaves of plants, such as spinach, castor bean, barley and Brassica have been conducted (see, Kang and Rawsthorne,
Plant J
6: 795-805 (1994); Miernyk and Dennis,
J. Exper. Bot.
34: 712-718 (1983); Smith et al.,
Plant Physiol.
98: 1233-1238 (1992); Liedvogel and Bauerle,
Planta
169: 481-489 (1986); Murphy and Leech, FEBS Letter 77: 164-168 (1977); Roughan et al.,
Biochem. J.
158: 593-601 (1976); Roughan et al.,
Biochem. J.
184: 565-569 (1978); Roughan et al.,
Biochem. J.
184: 193-202 (1979); Springer and Heise,
Planta
177: 417-421 (1989); Schulze-Siebert and Shultz,
Plant Physiol.
84: 1233-1237 (1987); and Heintze et al.,
Plant Physiol.
93: 1121-1127 (1990)). Such studies suggest that acetate is the preferred substrate for fatty acid synthesis in chloroplasts, while pyruvate is the preferred substrate for fatty acid synthesis in plastids in embryos.
The acetyl CoA so produced is then involved in fatty acid biosynthesis, i.e., the synthesis of the basic building blocks of membrane lipids, fats and waxes. A similar reaction is effected by mtPDH in the mitochondrion.
ACS is exclusively found in the plastids of plants and is strongly regulated by light (Sauer and Heise, Z.
Naturforsch
38 c: 399-404 (1983)). The amount of ACS is fairly constant between spinach, pea and amaranthus chloroplasts; there is about 20% more in corn chloroplasts. Given that the partially purified enzyme is completely DTT-dependent suggests that its activity in vivo may be regulated by the ferredoxin/thioredoxin system (Zeiher and Randall,
Plant Physiol.
96: 382-389 (1991)). There is some potential for weak feedback inhibition by acetyl CoA. The enzyme also has a high pH requirement, along with a dependency on a high ATP(Mg
2+
-ATP)/ADP ratio (Sauer and Heise (1983), supra). The ACS reaction should be substrate saturated because K
m
values for acetate are between 0.02 and 0.10 mM in spinach (Sauer and Heise (1983), supra; Zeiher and Randall (991), supra; and Treede and Heise, Z.
Naturforsch
40 c: 496-502 (1985)), peas (Treede and Heise (1985), supra), amaranthus (Roughan and Ohlrogge,
Anal. Biochem.
216: 77-82 (1 994)) and potatoes (Huang and Stumpf,
Arch Biochem. Biophys.
140: 158-173 (1970)), whereas the concentration of cellular acetate is estimated to be about 1.0 mM (Kuhn et al.,
Arch Biochem. Biophys.
209: 441-450 (1981)).
The pPDH appears to have the same general structure as mtPDH, being composed of a pyruvate dehydrogenase component (E1
&agr;
and E1
&bgr;
), a transacetylase component (E2), and dehydrolipoamide dehydrogenase (E3) subunits. The molecular weight of pPDH and its cofactor requirements are also similar to mtPDH, although affinities for NAD
+
and TPP vary somewhat (Camp and Randall,
Plant Physiol.
77: 571-577 (1985); Miernyk et al. (1983), supra; and Conner et al.,
Planta
200: 195-202 (1996)). pPDH, which is less sensitive to acetyl CoA than mtPDH, has an optimal pH of about 8.0 and requires about 10 mM Mg
2+
for maximal activity. While the activity of mtPDH is controlled by a sophisticated kinase/phosphatase system, which phosphorylates and thereby inactivates the E1
&agr;
subunit, pPDH is not subject to such regulation. However, pPDH is strongly regulated by the NADH/NAD
+
ratio and is moderately regulated by light. Regulation by ATP, NADPH, fatty acyl CoAs and glycolytic intermediates is minor (Camp et al.,
Biochim. Biophys. Acta
933: 269-275 (1988); and Qi et al.,
J. Exp. Bot.
47: 1889-1896 (1996)).
PDH activity varies from one tissue to the next with mtPDH activity varying 15-fold and pPDH activity varying 6-fold (Lernmark and Gardestrom,
Plant Physiol.
106: 1633-1638 (1994)). The ratio of pPDH/mtPDH also varies between plants, with 6.5 times more activity in the chloroplasts than in the mitochondria of wheat leaves to 6.7 times more activity in the mitochondria than in the chloroplasts of peas. Although chloroplasts have proportionally less PDH activity than mitochondria in pea as compared to wheat, the chloroplasts have nearly as much PDH as mitochondria in absolute terms.
ACL is an enzyme that is responsible for the generation of acetyl CoA. ACL generates acetyl CoA as follows:
citrate+ATP+CoASH→acetyl-CoA+oxaloacetate+ADP+P
i
,
wherein ADP represents adenosine diphosphate and P
i
represents orthophosphate and wherein the citrate is that which is generated in the TCA cycle in the mitochondrion.
The activity of ACL has been found to correlate with lipid accumulation in developing seeds of
Brassica napus
L. (Ratledge et al.,
Lipids
32(1): 7-12 (1997)) and in the supernatant of a developing soybean (
Glycine max
L. Merr., var. Harosoy 63) cotyledon homogenate (Nelson et al.,
Plant Physiol.
55: 69-72 (1975)). ACL also has been found in crude extracts from the endosperm tissue of germinating castor bean (
Ricinus communis
cv. Hale) and has been found to be maximally active in 4-5-day old seedlings (Fritsch et al.,
Plant Physiol.
63: 687-691 (1979)).
PDC is a cytosolic enzyme that is responsible for the generation of acetaldehyde from pyruvate. PDC generates acetaldehyde from pyruvate as follows:
pyruvate→acetaldehyde+CO
2
.
The acetaldehyde so produced can be acted upon by ALDH.
ALDH is responsible for the generation of acetate from acetaldehyde. ALDH generates acetate from acetaldehyde as follows:
acetaldehyde+NAD
+
+H
2
O→acetate+NADH
+
+H
+
.
The acetate so produced can then enter the plastids, where it can be converted to acetyl CoA through the action of ACS.
ACH is an enzyme that is known to exist in yeast and is believed to exist in the mitochondria of pla

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