Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-07-29
2002-02-19
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252300, C435S254200, C435S410000, C536S023200, C800S278000, C800S295000
Reexamination Certificate
active
06348339
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in degradation of branched-chain amino acids in plants and seeds.
BACKGROUND OF THE INVENTION
Amino acids, in addition to their role as protein monomeric units, are energy metabolites and precursors of many biologically important nitrogen-containing compounds, notably heme, physiologically active amines, glutathione, nucleotides, and nucleotide coenzymes. Excess dietary amino acids are neither stored for future use nor excreted. Rather they are converted to common metabolic intermediates such as pyruvate, oxaloacetate, and alpha-ketoglutarate. Consequently, amino acids are also precursors of glucose, fatty acids, and ketone bodies and are therefore metabolic fuels.
Hydroxymethylglutaryl-CoA lyase (EC 4.1.3.4), also called HMG-CoA lyase, is involved in the degradation of leucine, and participates in butanoate metabolism, and in the synthesis and degradation of ketone bodies. HMG-CoA lyase catalyzes the final step of ketogenesis and leucine catabolism in the mitochondrial matrix. The first reported HMG-CoA lyase gene was from
Pseudomonas mevalonii
(Anderson, D. H. and Rodwell, V. W. (1989)
J Bacteriol.
171:6468-6472). The active site of the
Pseudomonas mevalonii
HMG-CoA lyase has been identified. Cys-237 is required for catalysis (Hruz, P. W. et al. (1992)
Biochemistry
31:6842-6847). To date, HMG-CoA lyase has not been described in plants.
3-Hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) catalyzes the NAD-dependent, reversible oxidation of 3-hydroxbutyrate to methylmalonate (Rougraff, P. M. et al. (1989)
J. Biol. Chem.
264:5899-5903). In animals, it is a homodimeric mitochondrial protein involved in valine catabolism. In
Pseudomonas aeruginosa
(encoded by the mmsB), it is involved in the-distal valine metabolic pathway (Steele, M. I. et al. (1992)
J. Biol. Chem.
267:13585-13592). The sequence of 3-hydroxyisobutyrate dehydrogenase from eukaryotic and prokaryotic sources show that this enzyme has been well conserved throughout evolution. The pathway of valine catabolism ultimately leads to the production of succinyl-SCoA. Succinyl-SCoA can be converted to pyruvate via the TCA cycle and then to glucose. Thus, this enzyme is needed, along with several others in the catabolic pathway, to interconvert the carbon skeleton of valine into other useful metabolites. 3-hydroxyiso-butyrate dehydrogenase has not been isolated from plants yet, although rice ESTs encoding portions of this gene are present in the GenBank database.
Involved in the processing of leucine, isovalyryl-CoA dehydrogenase (EC 1.3.99.10) uses FAD to convert isovalyryl-CoA to beta-methylcrotonyl-CoA. This enzyme is found in the mitochondria and has similarity with other acyl-CoA dehydrogenases (long chain acyl-CoA (LCAD), short chain acyl-CoA (SCAD), and medium-chain (MCAD) acyl-CoA dehydrogenases). The structural relatedness of these enzymes suggests that they are members of a gene family that shares a common ancestral gene (Matsubara et. al. (1989)
J Biol Chem
264(27):16321-16331). Rice and oat ESTs exist in the GenBank database but the enzyme has not yet been isolated from plants.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding enzymes involved in degradation of branched-chain amino acids. Specifically, this invention concerns an isolated nucleic acid fragment encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase or an isovalyryl-CoA dehydrogenase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase or an isovalyryl-CoA dehydrogenase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding hydroxymethyl-glutaryl CoA oxidase, 3-hydroxyisobutyrate dehydrogenase or isovalyryl-CoA dehydrogenase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a branched-chain amino acid degradation enzyme selected from the group consisting of hydroxymethylglutaryl CoA oxidase, 3-hydroxyisobutyrate dehydrogenase or isovalyryl-CoA dehydrogenase.
In another embodiment, the instant invention relates to a chimeric gene encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of hydroxymethylglutaryl CoA oxidase, 3-hydroxyisobutyrate dehydrogenase or isovalyryl-CoA dehydrogenase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyisobutyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a hydroxymethylglutaryl CoA oxidase, a 3-hydroxyiso-butyrate dehydrogenase, or an isovalyryl-CoA dehydrogenase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of hydroxymethylglutaryl CoA oxidase, 3-hydroxyisobutyrate dehydrogenase or isovalyryl-CoA dehydrogenase in the transformed host cell; (c) optionally purifying the hydroxymethylglutaryl CoA oxidase, the 3-hydroxyisobutyrate dehydrogenase or the isovalyryl-CoA dehydrogenase expressed by the transformed host cell; (d) treating the hydroxymethylglutaryl CoA oxidase, the 3-hydroxyisobutyrate dehydrogenase or the isovalyryl-CoA dehydrogenase with a compound to be tested; and (e) comparing the activity of the hydroxymethylglutaryl CoA oxidase, the
Cahoon Rebecca E.
Hitz William D.
Kinney Anthony J.
Rafalski J. Antoni
Belopolsky, Esq. Inna Y.
E.I . du Pont de Nemours and Company
Slobodyansky Elizabeth
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