Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-05-14
2001-04-17
Campell, Bruce R. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S325000, C435S069100, C536S023100
Reexamination Certificate
active
06218528
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to polynucleotides and polypeptides of the MurB family, as well as their variants, hereinafter referred to as “MurB,” “MurB polynucleotide(s),” and “MurB polypeptide(s).”
BACKGROUND OF THE INVENTION
The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago,
Streptococcus pneumoniae
has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with
S. pneumoniae,
many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of
Streptococcus pneumoniae
infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate
Streptococcus pneumoniae
strains which are resistant to some or all of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery. The enzyme UDP-N-acetylenolpyruvylglucosamine reductase, encoded by the gene MurB catalyses the reduction of UDP-N-acetylpyruvylglucosamine to UDP-N-acetyl muramate, with the concomitant oxidation of NADPH. N-acetyl muramate is a precusor for peptidoglycan biosynthesis.
The gene has been sequenced from
Escherichia coli
(Pucci, M. J., Discotto, L. F. & Dougherty T. J., 1992, J. Bacteriol., 174, 1690-1693) and the enzyme over-expressed, purified and kinetically characterized (Benson, T. E., Marquardt, J. L., Marquardt, A. C., Etzkom, F. A. & Walsh, C. T. (1993) Biochemistry, 32, 2024-2030). There is also a crystal structure of this enzyme (Benson, T. E., Walsh, C. T., & Hogle, J. M., (1996) Structure, 4, 47-54).
The gene has also been sequenced from
Bacillus subtilis
and shown to be essential in this organism (Rowland et al., (1995) Gene 164, 113-116).
The discovery of a MurB homologue in the human pathogen
Streptococcus pneumoniae
will allow us to produce UDP-N-acetylenolpyruvylglucosamine reductase enzyme which can then be used to screen for novel antibiotics. Inhibitors of this enzyme will have utility in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there exists a need for polynucleotides and polypeptides, such as the MurB embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known MurB from
B. subtilis
protein.
SUMMARY OF THE INVENTION
The present invention relates to MurB, in particular MurB polypeptides and MurB polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting MurB expression or activity.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to MurB polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a MurB of
Streptococcus pneumoniae,
which is related by amino acid sequence homology to MurB from
B. subtilis
polypeptide. The invention relates especially to MurB having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4 respectively.
TABLE 1
MurB Polynucleotide and Polypeptide Sequences
(A) Streptococcus pneumoniae MurB polynucleotide sequence [SEQ ID NO:1].
5′-
AAGGTATTTTTATGGTCGTCAAATGTCTCTAAAAATGGTATAATGGAATGAATTTTGTAAAAGGAAGAGTGA
CATGTCTGTAAGAGAAAAAATGCTTGAAATCTTAGAAGGAATTGATATCCGTTTTAAGGAACCCTTGCATAG
CTATAGTTATACAAAAGTAGGTGGAGAGGCTGATTATTTGGTCTTTCCACGAAATCGTTTTGAGTTGGCTCG
CGTTGTGAAATTTGCCAACCAAGAAAATATCCCTTGGATGGTTCTTGGCAATGCAAGCAATATCATCGTTCG
TGATGGTGGGATTCGTGGATTTGTCATCTTGTGTGACAAGCTCAATAACGTTTCTGTTGATGGCTATACCAT
TGAAGCAGAAGCTGGGGCTAACTTGATTGAAACAACTCGCATTGCCCTCCGTCATAGTTTAACTGGCTTTGA
GTTTGCTTGTGGTATTCCAGGAAGCGTTGGCGGTGCTGTCTTTATGAATGCGGGTGCCTATGGTGGCGAGAT
TGCTCACATCTTGCAGTCTTGTAAGGTCTTGACCAAGGATGGAGAAATCGAAACCCTGTCTGCTAAAGACTT
GGCTTTTGGTTACCGCCATTCAGCTATTCAGGAGTCTGGTGCAGTTGTCTTGTCAGTTAAATTTGCCCTAGC
TCCAGGAACCCATCAGGTTATCAAGCAGGAAATGGACCGCTTGACGCACCTACGTGAACTCAAGCAACCTTT
GGAATACCCATCTTGTGGCTCGGTCTTTAAGCGTCCAGTCGGGCATTTTGCAGGTCAGTTAATTTCAGAAGC
TGGCTTGAAAGGCTATCGTATCGGTGGCGTAGAAGTGTCAGAAAAGCATGCAGGATTTATGATCAATGTCGC
AGATGGAACGGCCAAAGACTACGAGGACTTGATCCAATCGGTTATCGAAAAAGTCAAGGAACACTCAGGTAT
TACGCTTGAAAGAGAAGTCCGGATCTTGGGTGAAAGCCTATCGGTAGCGAAGATGTATGCAGGTGGTTTTAC
TCCCTGCAAGAGGTAGTGGGGACCTGACAGAGCCCCGATCGGTTAATCTATGAAAAAGAAGGAATTTATGCC
AATTGAAAAAACCAATTATCGA-3′
(B) Streptococcus pneumoniae MurB polypeptide sequence deduced from a
polynucleotide sequence in this table [SEQ ID NO:2].
NH
2
-
MSVREKMLEILEGIDIRFKEPLHSYSYTKVGGEADYLVFPRNRFELARVVKFANQENIPWMVLGNASNIIVR
DGGIRGFVILCDKLNNVSVDGYTIEAEAGANLIETTRIALRHSLTGFEFACGIPGSVGGAVFMNAGAYGGEI
AHILQSCKVLTKDGEIETLSAKDLAFGYRHSAIQESGAVVLSVKFALAPGTHQVIKQEMDRLTHLRELKQPL
EYPSCGSVFKRPVGHFAGQLISEAGLKGYRIGGVEVSEKHAGFMINVADGTAKDYEDLIQSVIEKVKEHSGI
TLEREVRILGESLSVAKMYAGGFTPCKR-COOH
(C) Streptococcus pneumoniae MurB ORF sequence [SEQ ID NO:3].
5′-
TAACGTTTCTGTTGATGGCTATACCATTGAAGCAGAAGCTGGGGCTAACTTGATTGAAACAACTCGCATTGC
CCTCCGTCATAGTTTAACTGGCTTTGAGTTTGCTTGTGGTATTCCAGGAAGCGTTGGCGGTGCTGTCTTTAT
GAATGCGGGTGCCTATGGTGGCGAGATTGCTCACATCTTGCAGTCTTGTAAGGTCTTGACCAAGGATGGAGA
AATC
Biswas Sanjoy
Brown James Raymond
Chalker Alison Frances
Holmes David John
Ingraham Karen A
Campell Bruce R.
Deibert Thomas S.
Gimmi Edward R.
King William T.
Shukla Ram R.
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