Phosphopantetheinyl transferases and uses thereof

Details Phosphopantetheinyl transferases and uses thereof Phosphopantetheinyl transferases and uses thereof

1996-10-11

2003-06-17

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

C435S069100, C435S193000, C435S068100, C536S023200, C536S023700

Reexamination Certificate

active

06579695

ABSTRACT:

BACKGROUND OF THE INVENTION
Acyl carrier protein (ACP) is a small acidic protein (8,800 Da) responsible for acyl group activation in fatty acid biosynthesis. The gene encoding ACP (acpP) has been cloned and overexpressed (Rawlings, M. and Cronan, J. E., Jr. (1992)
J. Biol. Chem.,
267, 5751-5754; Jones, A. L., et al. (1993)
Biochem. Soc. Trans.,
21, 202S) and the solution structure of ACP has been solved by NMR spectroscopy (Holak, T. et al. (1988)
Eur. J. Biochem.
175:9-15). Homologs of
E. coli
ACP exist throughout nature in two forms; either as an integral domain of a much larger multifunctional enzyme (type I) or as a discrete protein capable of associating with several other enzymes constituting a multienzyme synthase complex (type II). In these two forms, ACPs play central roles in a broad range of other biosynthetic pathways that depend on iterative acyl transfer steps, including polyketide (Shen, B., et al. (1992)
J. Bacteriol.
174:3818-3821), non-ribosomal peptide (Baldwin, J. E., et al. (1991)
J. Antibiot.
44:241-247), and depsipeptide biosynthesis (Rusnak, F., et al. (1991)
Biochemistry
30:2916-2927) as well as in the transacylation of oligosaccharides (Geiger, O., et al. (1991)
J. Bacteriol.
173:2872-2878) and proteins (Issartel, J. P., et al. (1991)
Nature
351:759-761).
A definitive feature of ACP is the 4′-phosphopantetheine (4′-PP) prosthetic group (Majerus, P. W. et al. (1965)
Proc. Natl. Acad. Sci. USA
53:410-417). 4′-PP is attached through a phosphodiester linkage to a conserved serine residue found in all ACPs. Acyl groups of the many substrates recognized by type I and type II ACPs are activated for acyl transfer through a thioester linkage to the terminal cysteamine thiol of the 4′-PP moiety. The &bgr;-alanyl and pantothenate portions of the 4′-PP structure are believed to serve as a tether between the phosphodiester-ACP linkage and the terminal thioester, suggesting that 4′-PP may function as a swinging arm, shuttling growing acyl chains between various active sites, e.g. as in the sequential addition of 11 amino acids by the 800 kDa cyclosporin synthetase (Lawen, A. and Zocher, R. (1990)
J. Biol. Chem.
265:11355-11360).
Holo-ACP synthase (holo-ACPS) transfers the 4′-PP moiety from Coenzyme A (CoA) to Ser-36 of apo-ACP to produce holo-ACP and 3′,5′-ADP in a Mg
2+
dependent reaction. The (acyl carrier synthase protein) ACPS from
E. coli
was partially purified 780-fold from crude extracts (Elovson, J. and Vagelos, P. R. (1968)
J. Biol. Chem.
243:3603-3611), and the ACPS from spinach has been partially purified (Elhussein, S. A., et al. (1988)
Biochem. J.
252:39-45), but remarkably little has been shown about the mechanism or specificity of this post-translational phosphopantetheinylation process. A mutant of
E. coli
conditionally defective in the synthesis of holo-ACP has been identified and the mutant phenotype attributed to an altered holo-ACP synthase activity (Polacco, M. L. and Cronan, J. E., Jr. (1981)
J. Biol. Chem.
256:5750-5754).
SUMMARY OF THE INVENTION
This invention pertains to isolated and purified natural and recombinant phosphopantetheinyl transferases, e.g., acyl carrier protein synthases (ACPSs), from eukaryotes, prokaryotes, or plants. Also within the scope of the invention are active fragments of phosphopantetheinyl transferases, modified phosphopantetheinyl transferases, and modified active fragments of phosphopantetheinyl transferases. These forms of phosphopantetheinyl transferase are preferably purified to at least about 60% purity, more preferably to at least about 70% purity, more preferably to at least about 80% purity, more preferably to at least about 90% purity and even more preferably to at least about 95% purity. The phosphopantetheinyl transferase of the invention can be used for in vitro phosphopantetheinylation of substrates, such as acyl carrier proteins (ACPs), which have, for example, been produced by overexpression in a host cell. Kits including the phosphopantetheinyl transferase described herein are also within the scope of the invention.
The invention also provides host cells modified to express at least one nucleic acid encoding at least one phosphopantetheinyl transferase or active fragment thereof. In one embodiment, the host cells of the invention are further modified to express at least one nucleic acid encoding at least one substrate of a phosphopantetheinyl transferase. Such host cells may further express nucleic acids encoding other components associated with the ACP. Modified host cells of the invention can be used for the production of antibiotics or other compounds whose synthesis requires an ACP.


REFERENCES:
patent: 5824513 (1998-10-01), Katz et al.
patent: 2100791 (1993-07-01), None
patent: WO 93/13663 (1993-07-01), None
Ratner et al., Biotechnology, vol 7, no 11, pp. 1129-1133, Nov. 1989.*
Plunkett (Oct. 23, 1995) GenBank accession AAA79825.*
Lambalot et al. A new enzyme superfamily—the phosphopantetheinyl transferases. Chem Biol. Nov. 1, 1996 vol. 3, No. 11, pp.: 923-936.*
Werkmeister et al. Coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase in yeast. Biochem Biophys Res Commun. Sep. 16, 1980, vol. 96, No. 1, 483-90.*
Gehring et al. Enterobactin biosynthesis inEscherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate. Biochemistry. Jul. 15, 1997, vol. 36, pp. 8495-8503.*
George et al, “Current methods in sequence comparison and analysis,” in Macromolecular Sequencing and Synthesis, Selected Methods and Applications, 1988, D.H. Schlesinger (ed.), Alan R. Liss, Inc., New York, NY, pp. 127-149.*
Barton, “Protein sequence alignment and database scanning,” in Protein Structure Prediction, A Practical Approach, 1996 IRL Press at Oxford University Press, Oxford, UK, pp. 31-63.*
Armstrong S. K. et al., “TheEscherichia colienterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product”,Molecular Microbiology, vol. 3(6) pp. 757-766 (1989).
Baldwin, Jack E. et al., “Isolation and Partial Characterisation of ACV Synthetase fromCephalosporium acremoniumandStreptomyces clavuligerus: Evidence for the Presence of Phophopantothenate in ACV Synthease”,The Journal of Antibiotics, vol. 44 No. 2 pp. 241-248 (1991).
Billich, Andrea et al., “Enzymatic Synthesis of Cyclosporin A*”,The Journal of Biological Chemistry, vol. 262 No. 36 pp 17258-17259 (1987).
Borchet, Stefan et al., “Induction of Surfactin Production inBacillus subtilisby gsp, a Gene Located Upstream of the Gramicidin S Operon inBacillus brevis”, Journal of Bacteriology, vol. 176 No. 8 pp. 2458-2462 (1994).
Coderre, Peter E. et al., “The entD Gene of theEscherichia coliK12 Enterobactin Gene Cluster”,Journal of General Microbiology, vol. 135 pp. 3042-3055 (1989).
Cosmina, Paola et al., “Sequence and anlysis of the genetic locus responsible for surfactin synthesis inBacillus subtilis”, Molecular Microbiology, vol. 8(5) pp. 821-831 (1993).
Elhussein, Salah A. et al., “Plant holo-(acyl carrier protein) synthase”,Biochemistry Journal, vol. 252 pp. 39-45 (1988).
Elovson, John et al., “Acyl Carrier Protein”,The Journal of Biological Chemistry, vol. 243 No. 13 pp. 3603-3611 (1968).
Fischl, Anthony S. et al., “Isolation and Properties of Acyl Carrier Protein Phosphodiesterase ofEscherichia coli”, Journal of Bacteriology, vol. 172 No. 9 pp. 5445-5449 (1990).
Geiger, Otto et al., “Isolation of theRhizobium leguminosarumNodF Nodulation Protein: NodF carries a 4′-Phosphopantetheine Prosthetic Group”Journal of Bacteriology, vol. 173 No. 9 pp. 2872-2878 (1991).
Gerngross, T. U. et al., “Overexpression and Purification of the Soluble Polyhydroxyalkanoate Synthase fromAlcaligenes eutrophus: Evidence for a Required Posttranslational Modification for Catalytic Activity”,Biochemistry, vol. 33 No. 31 pp. 9311-9320 (1994).
Grossman, Trudy H., et al., “Isolation and Characterization ofBacillus subtili

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