Aromatic amino acid catabolism enzymes

Details Aromatic amino acid catabolism enzymes Aromatic amino acid catabolism enzymes

1999-07-28

2001-04-17

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S183000, C435S189000, C435S190000, C536S023200

Reexamination Certificate

active

06218169

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 aromatic amino acid catabolism in plants and seeds.
BACKGROUND OF THE INVENTION
In addition to their role as protein monomeric units, amino acids are energy metabolites and precursors of many biologically important nitrogen-containing compounds, such as heme, physiologically active amines, glutathione, other amino acids, 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. The degradation of amino acids converts them to citric acid cycle intermediates or their precursors so that they can be metabolized to CO
2
and water or used in gluconeogenesis. Oxidative breakdown of amino acids typically accounts for 10 to 15% of the metabolic energy generated by animals.
The enzymes included in this application are involved in catabolism of the aromatic amino acids. The first reaction in phenylalanine degradation is its hydroxylation to tyrosine; thus a single pathway is responsible for the breakdown of both of these amino acids. 3,4-Dehydroxyphenyl acetate 2,3-dioxygenase is also called homogentisate 1,2-dioxygenase (EC 1.13.11.15) and, in the presence of oxygen, catalyzes the decyclization of homogentisic acid (3,4-dihydroxyphenylacetate) into 2-Hydroxy-5-carboxymethylmuconate semialdehyde. Loss of homogentisate 1,2 dioxygenase (HGO) activity is responsible for the human metabolic disorder alkaptonuria. A large number of variant forms of the human enzyme have been described which show the clinical effect of single nucleotide changes on the activity of the enzyme (Fernandez-Canon, J. M. et al. (1996).
Nat Genet
14:19-24). The gene
Aspergillus nidulans
homogentisate 1,2 dioxygenase has been characterized and its disruption shown to induce secretion of homogenistate ((1995)
J. Biol Chem
270:21199-21205). In the same article, the authors searched the GenBank database with the homogentisate 1,2 dioxygenase sequence and identified ESTs with significant similarity to homogentisate 1,2 dioxygenase. These ESTs were from tissues obtained from human,
Arabidopsis thaliana,
and
Ricinus communis.
Fumarylacetoacetase, also named fumarylacetoacetate hydrolase (EC 3.7.1.2) catalyzes the last step in the phenylalanine/tyrosine degradation catalyzing the conversion of 4-fumarylacetoacetate and water to acetoacetate and fumarate. Debilitating mutations in this enzyme have been shown to be the cause of hereditary tyrosinemia type I in humans (St-Louis, M. and Tanguay, R. M. (1997)
Hum. Mut.
9:291-299; Labelle, Y. et al. (1993)
Hum. Mol. Genet.
2:941-946).
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding enzymes involved in aromatic amino acid catabolism. Specifically, this invention concerns an isolated nucleic acid fragment encoding a homogentisate 1,2-dioxygenase or a fumarylacetoacetase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a homogentisate 1,2-dioxygenase or a fumarylacetoacetase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding homogentisate 1,2-dioxygenase or fumarylacetoacetase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an enzyme involved in aromatic amino acid catabolism selected from the group consisting of homogentisate 1,2-dioxygenase and fumarylacetoacetase.
In another embodiment, the instant invention relates to a chimeric gene encoding a homogentisate 1,2-dioxygenase or a fumarylacetoacetase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a homogentisate 1,2-dioxygenase or a fumarylacetoacetase, 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 homogentisate 1,2-dioxygenase or a fumarylacetoacetase, 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 homogentisate 1,2-dioxygenase or a fumarylacetoacetase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a homogentisate 1,2-dioxygenase or a fumarylacetoacetase; 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 homogentisate 1,2-dioxygenase or fumarylacetoacetase 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 homogentisate 1,2-dioxygenase or a fumarylacetoacetase.
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 homogentisate 1,2-dioxygenase or a fumarylacetoacetase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a homogentisate 1,2-dioxygenase or a fumarylacetoacetase, 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 homogentisate 1,2-dioxygenase or fumarylacetoacetase in the transformed host cell; (c) optionally purifying the homogentisate 1,2-dioxygenase or the fumarylacetoacetase expressed by the transformed host cell; (d) treating the homogentisate 1,2-dioxygenase or the fumarylacetoacetase with a compound to be tested; and (e) comparing the activity of the homogentisate 1,2-dioxygenase or the fumarylacetoacetase that has been treated with a test compound to the activity of an untreated homogentisate 1,2-dioxygenase or fumarylacetoacetase, thereby selecting compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
Enzymes Involved in Aromatic Amino Acid Catabolism
SEQ ID NO:
Protein
Clone Designation
(Nucleotide)
(Amino Acid)
Homogentisate 1,2-
cbn2.pk0052.e6
1
2
Dioxygenase
rls6.pk0027.h11
3
4
sfl1.pk0008.h2
5
6
wlk8.pk0020.a

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