Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
1999-10-04
2001-07-31
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S252300, C435S320100, C435S822000, C435S193000, C435S110000, C536S023200
Reexamination Certificate
active
06268188
ABSTRACT:
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been putatively identified as transaminases and/or aminotransferases. Aminotransferases are enzymes that catalyze the transfer of amino groups from &agr;-amino to &agr;-keto acids. They are also called transaminases.
The &agr;-amino groups of the 20 L-amino acids commonly found in proteins are removed during the oxidative degradation of the amino acids. The removal of the &agr;-amino groups, the first step in the catabolism of most of the L-amino acids, is promoted by aminotransferases (or transaminases). In these transamination reactions, the &agr;-amino group is transferred to the &agr;-carbon atom of &agr;-ketoglutarate, leaving behind the corresponding &agr;-keto acid analog of the amino acid. There is no net deamination (i.e., loss of amino groups) in such reactions because the &agr;-ketoglutarate becomes aminated as the &agr;-amino acid is deaminated. The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of only one, namely, L-glutamate. The glutamate channels amino groups either into biosynthetic pathways or into a final sequence of reactions by which nitrogenous waste products are formed and then excreted.
Cells contain several different aminotransferases, many specific for &agr;-ketoglutarate as the amino group acceptor. The aminotransferases differ in their specificity for the other substrate, the L-amino acid that donates the amino group, and are named for the amino group donor. The reactions catalyzed by the aminotransferases are freely reversible, having an equilibrium constant of about 1.0 (&Dgr;G
0′
≃0 kJ/mol).
Aminotransferases are classic examples of enzymes catalyzing bimolecular ping-pong reactions. In such reactions the first substrate must leave the active site before the second substrate can bind. Thus the incoming amino acid binds to the active site, donates its amino group to pyridoxal phosphate, and departs in the form of an &agr;-keto acid. Then the incoming &agr;-keto acid is bound, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid.
The measurement of alanine aminotransferase and aspartate aminotransferase levels in blood serum is an important diagnostic procedure in medicine, used as an indicator of heart damage and to monitor recovery from the damage.
The polynucleotides and polypeptides of the present invention have been identified as transaminases and/or aminotransferases as a result of their enzymatic activity.
In accordance with one aspect of the present invention, there are provided novel enzymes, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the enzymes of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such enzymes.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said enzymes and subsequent recovery of said enzymes.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes for transferring an amino group from an ax-amino acid to an &agr;-keto acid. Most transaminases use L-amino acids as substrates, but as described below, it is also possible to convert the transaminases of the invention to use D-amino acids as substrates, thereby increasing their array of uses to include, for example, manufacture of synthetic pyrethroids and as components of &bgr;-lactam antibiotics. The transaminases of the invention are stable at high temperatures and in organic solvents and, thus, are superior for use with L- and/or D-amino acids for production of optically pure chiral compounds used in pharmaceutical, agricultural and other chemical industries.
In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process, for utilizing such enzymes, or polynucleotides encoding such enzymes, for in vitro purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar enzymes from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
FIG. 1
is an illustration of the full-length DNA (SEQ ID NO: 17) and corresponding deduced amino acid sequence (SEQ ID NO:25) of Aquifex aspartate transaminase A of the present invention. Sequencing was performed using a 378 automated DNA sequencer (Applied Biosystems, Inc.) for all sequences of the present invention.
FIG. 2
is an illustration of the full-length DNA (SEQ ID NO:18) and corresponding deduced amino acid sequence (SEQ ID NO:26) of Aquifex aspartate aminotransferase B.
FIG. 3
is an illustration of the full-length DNA (SEQ ID NO:19) and corresponding deduced amino acid sequence (SEQ ID NO:27) of Aquifex adenosyl-8-amino-7-oxononanoate aminotransferase.
FIG. 4
is an illustration of the full-length DNA (SEQ ID NO:20) and corresponding deduced amino acid sequence (SEQ ID NO:28) of Aquifex acetylornithine aminotransferase.
FIG. 5
is an illustration of the full-length DNA (SEQ ID NO:21) and corresponding deduced amino acid sequence (SEQ ID NO:29) of
Ammonifex degensii
aspartate aminotransferase.
FIG. 6
is an illustration of the full-length DNA (SEQ ID NO:22) and corresponding deduced amino acid sequence (SEQ ID NO:30) of Aquifex glucosamine:fructose-6-phosphate aminotransferase.
FIG. 7
is an illustration of the full-length DNA (SEQ ID NO:23) and corresponding deduced amino acid sequence (SEQ ID NO:31) of Aquifex histidinolphosphate aminotransferase.
FIG. 8
is an illustration of the full-length DNA (SEQ ID NO:24) and corresponding deduced amino acid sequence (SEQ ID NO:32) of
Pyrobacullum aerophilum
branched chain aminotransferase.
FIG. 9
is an illustration of the full-length DNA (SEQ ID NO:35) and corresponding deduced amino acid sequence (SEQ ID NO:36) of Ammonifex histidinol phosphate aminotransferase.
FIG. 10
is an illustration of the full-length DNA (SEQ ID NO:39) and corresponding deduced amino acid sequence (SEQ ID NO:40) of Aquifex aspartate aminotransferase.
FIG. 11
is a diagramatic illustration of the assay used to assess aminotransferase activity of the proteins using glutamate dehydrogenase.
The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
A coding sequence is “operably lined to” another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences ultimately process to produce the desired protein.
“Recombinant” enzymes refer to enzy
Swanson Ronald V.
Warren Patrick V.
Diversa Corporation
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Slobodyansky Elizabeth
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