Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1999-02-02
2002-07-02
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C514S017400, C530S812000
Reexamination Certificate
active
06413732
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to substrate analogs of UDP-GlcNAc:muramyl pentapeptide pyrophosphoryl, N-acetylglucosaminyltransferase (GlCNAc transfer, MurG, or its homologs), an enzyme involved in a bacterial cell wall biosynthesis. The substrate analogs of the invention are useful as functional substitutes of Lipid I, the membrane bound, natural substrate of MurG. In particular, the substrate analogs of the present invention can be used advantageously in an assay for the enzymatic activity catalyzed by MurG, in methods for identifying other substrate analogs of MurG, as well as inhibitors of enzymatic activity or cell wall biosynthesis (i.e., potential antibacterial drugs), and in the isolation/purification of MurG, including studies of its active protein/peptide fragments.
2. BACKGROUND OF THE INVENTION
2.1 Bacterial Enzymology
The emergence of resistance to existing antibiotics has rejuvenated interest in bacterial enzymology. It is hoped that detailed mechanistic and structural information about bacterial enzymes involved in critical biosynthetic pathways could lead to the development of new antibacterial agents. Because interference with peptidoglycan biosynthesis is a proven strategy for treating bacterial infections, all of the enzymes involved in peptidoglycan biosynthesis are potential targets for the development of new antibiotics. While some detailed structural and mechanistic information on some of the early enzymes in the pathway is now available, most of the downstream enzymes have proven very difficult to study.
There are two main reasons for this difficult: First, the downstream enzymes are membrane-associated, making them intrinsically hard to handle; secondly, discrete substrates for most of the downstream enzymes are either not available or not readily so. In some cases monomeric substrates are difficult to obtain in large quantities from natural sources. In other cases substrates, which may be available in large quantities from natural sources, are intractable polymeric substances. In the absence of readily available discrete substrates, it has been impossible to develop enzyme assays that can be used to measure the activity of the downstream enzymes reliably and under a well-defined set of reaction conditions. This unfulfilled need has thwarted attempts to purify many of the downstream enzymes in an active form suitable for structural characterization, much less permitted attempts to obtain detailed mechanistic information on such enzymes.
Some of the best antibiotics function by interfering with the biosynthesis of the peptidoglycan polymer that surrounds bacterial cells. With the emergence of bacterial pathogens that are resistant to common antibiotics it has become imperative to learn more about the enzymes involved in peptidoglycan biosynthesis. Although remarkable progress has been made in characterizing some of the early enzymes in the biosynthetic pathway (See, e.g., (a) Fan, C.; Moews, P. C.; Walsh, C. T.; Knox, J. R. Science 1994, 266, 439; (b) Benson, T. E.; Filman, D. J.; Walsh, C. T.; Hogle, J. M. Nat. Struct. Biol. 1995, 2, 644; (c) Jin, H. Y.; Emanuele, J. J.; Fairman, R.; Roberston, J. G.; Hail, M. E.; Ho, T.; Falk, P.; Villafranca, J. J. Biochemistry 1996, 35, 1423; (d) Skarzynski, T.; Mistry, A.; Wonacott, A.; Hutchinson, S. E.; Kelly, V. A.; Duncan, K. Structure 1996, 4, 1465; (e) Schonbrunn, E.; Sack, S.; Eschenburg, S.; Perrakis, A.; Krekel, F.; Amrhein, N.; Mandelkow, E. Structure 1996, 4, 1065. (f) Benson, T. E.; Walsh, C. T.; Hogle, J. M. Biochemistry 1997, 36, 806.), the downstream enzymes have proven exceedingly difficult to study. Part of the difficulty steams from the fact that such downstream enzymes are membrane-associated (See, e.g., (a) Gittins, J. R.; Phoenix, D. A.; Pratt, J. M. FEMS Microbiol, Rev. 1994, 13, 1; (b) Bupp, K.; van Heijenoort, J. 1993, 175, 1841.), making them intrinsically hard to handle, and partly because substrates for many of the enzymes are not readily available. (See, e.g., (a) Pless, D. D.; Neuhaus, F. C. J. Biol. Chem. 1973, 248, 1568; (b) van Heijenoort, Y.; Gomez, M.; Derrien, M.; Ayala, J.; van Heijenoort, J. J. Bacterial, 1992, 174, 3549.) These problems have impeded the development of activity assays suitable for detailed mechanistic investigations of the downstream enzymes. For a fluorescent assay to monitor Mra Y activity, see: Brandish, P. E.; Burnham, M. K.; Lonsdale, J. T.; Southgate, R.; Inukai, M.; Bugg, T. D. H. J. Biol. Chem. 1996, 271, 7609.
2.2. MurG
One such downstream enzyme is MurG, which is involved in peptidoglycan biosynthesis. MurG catalyzes that last intracellular step in the biosynthetic pathway of peptidoglycan biosynthesis, i.e., the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) to the lipid-linked N-acetylmuramylpentapeptide substrate, Lipid I. (See, Scheme 1, below.)
Although the murG gene is first identified in
E. coli
in 1980 and is sequenced independently by two groups in the early 1990's, very little is known about the MurG enzyme. There are no mammalian homologs, and no direct assays for MurG activity have been developed, in part because the lipid-linked substrate (Lipid I, Scheme 1) is extremely difficult to isolate. This lipid-linked substrate is present only in minute quantities in bacterial cells. Although it is possible to increase the quantities of lipid-linked substrate by using cells engineered to overexpress enzymes involved in the synthesis of the lipid-linked substrate, isolation remains very difficult. Moreover, the isolated substrate is hard to handle.
Consequently, MurG activity is currently assessed using crude membrane preparations by monitoring the incorporation of radiolabel from radiolabeled UDP-GlcNAc donor group into lipid-linked acceptor components in the membrane. To increase the signal, the membranes are often prepared from bacterial cultures that overexpress MraY and/or MurG. MraY is the enzyme that catalyzes the reaction that attaches the MraY substrate, UDP-N-acetyl muramic acid pentapeptide, to a lipid phosphate moiety to provide Lipid I, which is the substrate for MurG. Typically, the membrane preparations are supplemented with exogenous UDP-N-acetyl muramic acid pentapeptide for conversion to Lipid I. This MraY substrate can be readily isolated in large quantities from bacterial cultures. Although this “coupled” enzyme assay is manageable for screening of potential inhibitors of the MurG enzyme, it is not suitable for detailed mechanistic investigations, and it cannot be used to follow MurG activity during purification.
More specifically, MurG is a cytoplasmic membrane-associated enzyme which catalyzes the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) to the C4 hydrozyl of an undecaprenyl pyrophosphate N-acetylmuramyl pentapeptide substrate (Lipid I). resulting in the assembly of the disaccharide-pentapeptide building block (Lipid II, Scheme 1), which is incorporated into polymeric peptidoglycan. See, e.g., (a) Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 199; (b) Mengin-Lecreulx, D.; Fluoret, B.; van Heijenoort, J. . Bacterial. 19R2, 151, 1109. As already mentioned, the muramyl pentapeptide substrate is unique to bacteria. Hence, the MurG enzyme is a potential target for the discovery or design of specific MurG inhibitors.
Despite decades of effort spent characterizing MurG activity, there is virtually no structural or mechanistic information on the enzyme. See, e.g., (a) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, J. L. Proc. Natl. Acad. Sci. USA 1965, 53, 881; (b) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, . L. J. Biol. Chem. 1967, 242, 180; (c) Taku, A.; Fan, D. P. J. Biol. Chem. 1976, 251, 6154; (d) Mengin-Lecreulx, D.; Texier, L.; van Heijenoort, J. Nucl. Acid. Res. 1990, 18, 2810; (e) Ikeda, M.; Wachi, M.; Jung, H. K.; Ishino, F.; Matsuhashi, M. Nucl. Acid Res. 1990, 18, 4014; (f) Mengin-Lecreulx, D.; Texier, L.; Rousseau, M.; van Heijenoort, J. J. Bacteriol 1991, 173, 4652; (g) Miyao, A.; Yoshimura, A.; Sato, T.; Yamamoto, T.; Theeragool, T.; Kobayash
Ge Min
Kahne Suzanne Walker
Men Hongbin
Park Peter
Meller Mike
Naff David M.
The Trustees of Princeton University
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