Muri protein from Streptococcus pneumoniae

Details Muri protein from Streptococcus pneumoniae Muri protein from Streptococcus pneumoniae

1999-04-30

2001-01-09

Navarro, Albert (Department: 1645)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C435S233000, C530S350000, C530S820000, C530S825000

Reexamination Certificate

active

06171834

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of the murI gene encoding glutamate racemase of
Streptococcus pneumoniae
and the use of the murI gene and the encoded protein in a screen for new inhibitors of bacterial cell wall biosynthesis.
The emergence of antibiotic resistance in common pathogenic bacterial species has justifiably alarmed the medical and research communities. Frequently these organisms are co-resistant to several different antibacterial agents. Pathogens resistant to frequently utilized antibiotics are found in the clinical as well as the community setting. Particularly problematic in the community setting has been the emergence and rapid spread of beta-lactam resistance in
Streptococcus pneumoniae
which frequently causes upper respiratory tract infections. Resistance to beta-lactams in this organism is due to modification of one or more of the penicillin-binding proteins (PBP's) which are involved in cell wall biosynthesis and are the targets for beta-lactam antibiotics.
Interference with bacterial cell wall biosynthesis is an especially attractive antibacterial target because an analogous structure does not exist in mammalian cells so that compounds that interfere with cell wall biosynthesis have low toxicity in humans and potentially high therapeutic value.
The bacterial cell wall structure contains a peptidoglycan layer which provides mechanical rigidity for the bacterium. This segment of the cell wall is composed of a sugar backbone (alternating residues of N-acetylglucosamine and N-acetylmuramic acid) attached to a pentapeptide (also referred to as “stem peptide,” or “Park nucleotide”) containing alternating D and L amino acid residues. The nascent peptidoglycan layer is stabilized by an enzymatic step which crosslinks adjacent pentapeptide moieties. Without this crosslinking step the peptidoglycan structure is severely weakened and susceptible to degradation. Indeed, it is the peptidoglycan crosslinking step that has been a frequently targeted site for antibiotic compounds such as the beta-lactam antibiotics.
Unlike the peptidoglycan crosslinking step, the stem peptide pathway has not been widely exploited as a target for inhibitory compounds. The stem peptide biosynthetic pathway comprises at least 10 steps in which the stem peptide is added onto UDPMurNAc by the stepwise addition of amino acid residues. In the first step, catalyzed by the UDPGlcNAc enolpyruvyl transferase and NADH-dependent reductase, UDPGlcNAc is converted to UDPMurNAc. In five subsequent steps, catalyzed by UDP-N-acetylmuramate:L-alanine ligase; UDP-N-acetyl-muramyl-L-alanine:D-glutamate ligase; UDP-N-acetyl-muramyl-L-alanyl-D-isoglutamate:L-lysine ligase; UDP-N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysine:D-alanyl-D-alanine ligase; and D-alanyl-D-alanine ligase, the final product, UDPMurNAc-L-Ala-D-isoGlu-L-lysine-D-Ala-D-Ala, is produced in
Streptococcus pneumoniae.
The enzymatic steps involved in the formation of the stem peptide are potential targets for new antibacterial agents. A few inhibitors, which target this pathway, have been developed. For example, D-cycloserine inhibits alanine racemase and D-alanine-D-alanine ligase; phosphonomycin inhibits the conversion of UDP-GlcNAc to UDP-GlcNac-enolpyruvate; and Alafosfalin inhibits the formation of UDP-MurNac-L-Ala.
While inroads in the development of new antibiotics and new targets for antibiotic compounds have emerged in a variety of microorganisms, progress has been less apparent in
Streptococcus pneumoniae.
In part,
Streptococcus pneumoniae
presents a special case because this organism is highly mutagenic and readily takes up and integrates exogenous foreign DNA from its surroundings, thereby increasing the possibility of acquiring or creating novel genes. Thus, the need for new antibacterial compounds and new targets for antibacterial therapy is especially acute in
Streptococcus pneumoniae.
SUMMARY OF THE INVENTION
The present invention is designed to meet the aforementioned need and provides, inter alia, isolated nucleic acid molecules that encode the murI gene product from
Streptococcus pneumoniae.
The invention also provides the protein product of the
Streptococcus pneumoniae
murI gene, glutamate racemase (MurI protein), in substantially purified form.
Having the cloned murI gene of
Streptococcus pneumoniae
enables the production of recombinant MurI protein and the implementation of large scale screens to identify new inhibitory compounds targeted at the stem peptide biosynthetic pathway. It may be possible to combine stem peptide biosynthetic enzymes in a single screen to examine several steps at the same time. Structural analysis of the MurI protein will enable structure-based drug design to develop novel compounds effective in the treatment of antibiotic resistant microorganisms.
In one embodiment the present invention relates to an isolated DNA molecule encoding MurI protein, said DNA molecule comprising the nucleotide sequence identified as SEQ ID NO. 1:
In another embodiment the present invention relates to a MurI protein molecule, wherein said protein molecule comprises the sequence identified as SEQ ID NO. 2.
In a further embodiment the present invention relates to a ribonucleic acid molecule encoding MurI protein, said ribonucleic acid molecule comprising the sequence identified as SEQ ID NO. 3:
In yet another embodiment, the present invention relates to a recombinant DNA vector which incorporates the
Streptococcus pneumoniae
murI gene in operable linkage to gene expression sequences enabling the murI gene to be transcribed and translated in a host cell.
In still another embodiment the present invention relates to homologous or heterologous host cells which have been transformed or transfected with the cloned murI gene of
Streptococcus pneumoniae
such that the murI gene is expressed in the host cell.
In a still further embodiment, the present invention relates to a method for identifying compounds that inhibit the enzymatic activity of the MurI protein of
Streptococcus pneumoniae.


REFERENCES:
patent: 5681694 (1997-10-01), Hoskins et al.
patent: 5712108 (1998-01-01), Peery et al.
patent: 5802875 (1991-03-01), Lacks et al.
Doublet, et al.,Biochemistry, 33:17, pp. 5285-5290, (1994).
Pucci, et al.,J. of Bacteriology, 176:2, pp. 528-530, (1994).
Baliko G. and Venetianer P.,J. of Bacteriology, 175:20, pp. 6571-6577, (1993).
Doublet, et al.,J. of Bacteriology, 174:10, pp. 2970-2979, (1993).
Doublet, et al.,J. of Bacteriology, 174:18, pp. 5772-5779 (1992).
Yoshimura, et al.,J. of Biological Chemistry, 268:32, pp. 24242-24246, (Nov. 15, 1993).
Burgess, et al.,J. of Cell Biology, vol. 111, pp. 2129-2138, (Nov. 1990).
Lazar, et al.,Molecular and Cellular Biology, pp. 1247-1252, (Mar. 1988).
Gallo, et al.,Biochemistry, 32:13, pp. 3981-3990, (1993).
Pucci, et al.,J. of Bacteriology, vol. 177, pp. 336-342, (Jan. 1995).
Nakajima, et al.,Agric. Biol. Chem., 52:12, pp. 3099-3104, (1988).
Viering, et al.,J. of Infectious Diseases, 160:1, pp. 76-82, (Jul. 1989).

Affiliated with

Also associated with

Rating

Muri protein from Streptococcus pneumoniae does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Muri protein from Streptococcus pneumoniae, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Muri protein from Streptococcus pneumoniae will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2508823