Murd protein and gene of Streptococcus pyogenes

Details Murd protein and gene of Streptococcus pyogenes Murd protein and gene of Streptococcus pyogenes

2000-05-03

2004-06-08

Bugaisky, Gabriele (Department: 1653)

Chemistry: molecular biology and microbiology

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

C435S069100, C435S320100, C435S252300, C435S254110, C536S023200

Reexamination Certificate

active

06746858

ABSTRACT:

STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
1. Field of the Invention
This invention relates to the genes and enzymes involved in cell wall synthesis in bacteria, and particularly to the inhibition of such enzymes.
2. Background of the Invention
The molecular target of many naturally-occurring antibiotics, including fosfomycin, cycloserine and b-lactams, is the synthesis of the bacterial cell wall. The frequency with which these types of antibiotics arose in evolution indicates that the pathway of cell wall biosynthesis is a particularly effective point of attack against bacteria. Genetic studies confirm the soundness of this process as a target, as temperature-sensitive alleles of the intracellular pathway genes are lytic, and therefore lethal. Since the building blocks of the cell wall are highly conserved structures in both Gram-positive and Gram-negative bacteria, but are unique to the eubacteria, novel inhibitors of cell wall formation are expected to be both broad spectrum and safe antibiotics.
The bacterial cell wall is a polymer—a single molecule composed of peptidoglycan—that defines the boundary and shape of the cell. Assembled by crosslinking glycan chains with short peptide bridges (Rogers, H. J., H. R. Perkins, and J. B. Ward, 1980, Biosynthesis of peptidoglycan. p. 239-297. In Microbial cell walls and membranes. Chapman & Hall Ltd. London), the completed structure is strong enough to maintain cell integrity against an osmotic pressure differential of over four atmospheres, but also flexible enough to allow the cell to move, grow and divide.
The construction of the peptidoglycan begins in the cytoplasm with an activated sugar molecule, UDP-N-acetylglucosamine. After two reactions (catalyzed by MurA and MurB) that result in the placement of a lactyl group on the 3-OH of the glucosamine moiety, a series of ATP-dependent amino acid ligases (MurC, -D, -E, and -F) catalyze the stepwise synthesis of the pentapeptide sidechain using the newly synthesized lactyl carboxylate as the first acceptor site. After attachment of the sugar pentapeptide to a lipid carrier in the plasma membrane, another glucosamine unit is added to the 4-OH of the muramic acid moiety. The completed monomeric building block is moved across the membrane into the periplasm where the penicillin-binding proteins enzymatically add it into the growing cell wall.
Because the pentapeptide sidechain is not synthesized ribosomally it contains more diverse chemical functionality than a typical peptide, both structurally and stereochemically. Two of the enzymes catalyze the addition of D-amino acids (MurD and MurF) and MurE mediates the formation of a peptide bond between the g-carboxylate of D-glutamate and the amino group of L-lysine. Presumably these structures render the exposed peptidoglycan resistant to the action of proteases, but they also imply that the active sites of the enzymes must have unusual structures in order to handle the somewhat uncommon substrates. These unusual active sites are targets to bind novel inhibitors that can have antimicrobial activity.
Among these potential enzyme targets is MurD. The first partial purification and characterization of a D-glutamate-adding enzyme was from
Staphlococcus aureus
(Ito, E. and J. L. Strominger, 1962, Enzymatic synthesis of the peptide in bacterial uridine nucleotides: Enzymatic addition of L-alanine, D-glutamic acid, and L-lysine. J. Biol. Chem. 237: 2689-2695; Nathenson, S. G., J. L. Strominger, and E. Ito, 1964, Enzymatic synthesis of the peptide in bacterial uridine nucleotides: purification and properties of D-Glutamic acid-adding enzyme, J. Biol. Chem. 239: 1773-177), followed by studies in more detail on the isolated
E. coli
enzyme (Blanot, D., A. Kretsovali, M. Abo-Ghalia, D. Mengin-Lecreulx, and J. van Heijenoort, 1983. Synthesis of analogues of precusors of bacterial peptidoglycan. p. 311-314. In Peptides. Blaha, K. and P. Malon, eds. pp. 311-314, Walter de Gryter and Co. Berlin, N.Y.; Jin, H., Emanuele, J. J., Jr., Fairman, R., Robertson, J. G., Hail, M. E., Ho, H.-T., Falk, P. and Villafranca, J. J., 1996. Structural studies of
Escherichia coli
UDP-N-acetylmuramate: L-alanine ligase. Biochemistry 35: 14423-14431; Ito E. and J. L. Strominger, 1973. Enzymatic synthesis of the peptide in bacterial uridine nucleotides: Comparative biochemistry. J. Biol. Chem. 248: 3131-3136; Michaud, C. D. Blanot, B. Flouret, and J. van Heijenoort, 1987. Partial purification and specificity studies of the D-glutamate-adding and D-alanyl-D-alanine-adding enzymes from
Escherichia coli
K12. Eur. J. Biochem. 166: 631-637). Recently, a purified recombinant
E. coli
MurD was reported (Pratviel-Sosa F, D. Mengin-Lecreulx and J. van Heijenoort, 1991. Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligase from
Escherichia coli.
Eur. J. Biochem. 202 (3):1169-1176) and genes encoding MurD have been cloned from several species of bacteria including
E. coli
(Ikeda, M., M. Wachi, F. Ishino, and M. Matsuhashi, 1990, Nucleotide sequence involving murD and an open reading frame ORF-Y spacing murF and ftsW in
Escherichia coli.
Nucleic Acids Res. 18:1058; Mengin-Lecreulx, D., C Parquet, L. Desviat, J. Pla, B. Flouret, J. Ayala and J. van Heijenoort, 1989, Organization of the murE-murG region of
Escherichia coli:
Identification of the murD gene encoding the D-glutamic-acid-adding enzyme. J. Bacteriol. 171: 6126-6134) and
B. subtilis
(Daniel, R. A., and J. Errington, 1993, DNA sequence of the murE-murD region of
Bacillus subtilis
168. J. Gen. Microbiol. 139:361-370; Henriques, A. O. de Lencaster, H. and P. J. Piggot, 1992, A
Bacillus subtilis
morphogene cluster that includes spoVE is homologous to the mra region of
Escherichia coli.
Biochimie. 74: 735-748). Compounds have been designed and synthesized that have inhibitory activity against the
E. coli
enzyme (Tanner, M. E., S. Vaganay, van Heijenoort, J., and D. Blanot, 1996, Phosphinate Inhibitors of the D-Glutamic Acid-Adding Enzyme of Peptidoglycan Biosynthesis. J. Org. Chem. 61: 1756-1760), although they do not have antibacterial activity.
SUMMARY OF THE INVENTION
Polynucleotides and polypeptides of
Streptococcus pyogenes
MurD, an enzyme involved in bacterial cell wall biosynthesis are provided. The recombinant MurD enzyme is catalytically active in ATP-dependent D-glutamate addition reactions. The enzyme is used in in vitro assays to screen for antibacterial compounds that target cell wall biosynthesis. The invention includes the purified polynucleotides, purified enzymes encoded by the polynucleotides, and host cells expressing the recombinant enzyme and their use in assays.


REFERENCES:
patent: 5681694 (1997-10-01), Hoskins et al.
Mengin-Lecreulx, D. et al., “Nucleotide sequence of the murD gene . . . synthetase ofEscherichia coli” Nucleic Acids Research 18, 1990, p. 183.
Michaud, C. D. Blanot, et al., “Partial Purification and specificity studies of the D-glutamate-adding . . . fromEscherichia coli” Eur J. Biochem 166, 1987, pp. 631-637.
Michaud, C., et al., “Revised Interpretation of the sequence containing the murE gene . . . synthease ofEscherichia coli” Biochem J. 269, 1990, pp. 277-280.
Nathenson, S. G., et al. “Enzymatic synthesis of the peptide in bacterial uridine nucleotides: purification and properties of D-Glutamic acid-adding enzyme” J. Biol. Chem. 239, 1964, pp. 1773-1776.
Pratviel-Sosa, F. et al., Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligase fromEscherichia coli. Eur J. Biochem 202(3), 1991, pp. 1169-1176.
Fervetti et al. Complete genome Sequence of an MI Strain ofStreptococcus pyogenes. Proc. Nat. Acad. Sci, USA. 2001 98(8):4658-4663.
Rogers, H.J., et al., “Biosynthesis of peptidoglycan” In Microbial Cell Walls and Membranes, 1980 Chapman & Hall Ltd. London pp. 239-297.
Schleifer, K. H. et al. “Peptidoglycan types of bacterial cell walls and their taxonomic implications”, Bacterio

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