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The Monte Ahuja College of Business Administration is part of Cleveland State University, in Cleveland, Ohio. Cleveland State University is a public institution of higher education in downtown Cleveland, Ohio. While every effort has been made to follow citation style rules, there may be some discrepancies. George et al, “Current methods in sequence comparison and analysis,” in Macromolecular Sequencing and Synthesis, Selected Methods and Applications, 1988, D.H. Barton, “Protein sequence alignment and database scanning,” in Protein Structure Prediction, A Practical Approach, 1996 IRL Press at Oxford University Press, Oxford, UK, pp.
Billich, Andrea et al., “Enzymatic Synthesis of Cyclosporin A*”, The Journal of Biological Chemistry, vol. Borchet, Stefan et al., “Induction of Surfactin Production in Bacillus subtilis by gsp, a Gene Located Upstream of the Gramicidin S Operon in Bacillus brevis”, Journal of Bacteriology, vol. Cosmina, Paola et al., “Sequence and anlysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis”, Molecular Microbiology, vol. Issartel, Jean-Paul et al., “Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation”Nature, vol.
Jackowski, Suzanne et al., “Metabolism of 4?-Phosphopantetheine in Escherichia coli”, Journal of Bacteriology, vol.
Lam, Hon-Ming et al., “Suppression of Insertions in the Complex pdxJ Operon of Escherichia coli K-12 by Ion and Other Mutations”, Journal of Bacteriology, vol. Lawen, Alfons et al., “Cell-Free Biosynthesis of New Cyclosporins”, The Journal of Antibiotics, vol.
Perego, Marta et al., “Incorporation of D-Alanine into Lipoteichoic Acid and Wall Teichoic Acid in Bacillus subtilis”, The Journal of Biological Chemistry, vol. Rawlings, Merriann et al., “The Gene Encoding Escherichia coli Acyl Carrier Protein Lies within a Cluster of Fatty Acid Biosynthetic Genes*”, The Journal of Biological Chemistry, vol.
Shen, Ben et al., “Purification and Characterization of the Acyl Carrier Protein of the Streptomyces glaucescens Tetracenomycin C Polyketide Synthase”, Journal of Bacteriology, vol. Stein, Torsten et al., “Gramicidin S Synthetase 1 )Pheylalanine Racemase), a Protype of Amino Acid Racemases Containing the Cofactor 4?-Phosphopantetheine”, Biochemistry, vol. Ullrich, Christian et al., “Cell-Free Biosynthesis of Surfactin, a Cyclic Lipopeptide Produced by Bacillus subtilis”, Biochemisstry, vol. Savage et al., “Phosphopantethenylated precursor acly carrier protein is imported into spinach (spinacia oleracea) chloroplasts,” Plant Physiol.
GOVERNMENT SUPPORTWork described herein was supported in part by NIH grants GM20011 and GM16583-02. Studying abroad is a remarkable idea first pioneered at Indiana University in the 1870s as a summer trip program to places like Switzerland, Germany, France, or Italy. Nowadays many accredited institutes from across the globe have study abroad and exchange programs to facilitate cultural exchange. For the students, international programs are important components of education, allowing students to experience different cultures and perspectives that they would otherwise not encounter or only encounter passively. Out in the wide, wide world, students will meet lots of different people of all kinds, but in general, students travelling abroad will encounter certain types of people.
Whatever the situation though, be it being lost in a city, unable to understand a transaction, or just stumbling with the language, someone will take pity on your poor self and help you out. They may show you where the best restaurants are, where to go shopping, when is best to get work done. Your mutual stumbling through the local language will help you help each other learn and grow in a mutually confusing and difficult time. It was established in 1964, inheriting the faculty, student body, and buildings of Fenn College, which had been founded in 1929.
Both the presursor and mature forms are substrates for phosphopantetheine attachment by a soluble chloroplast holo-acyl carrier protein synthase,” J. A method for phosphopantetheinylating a substrate in a cell or increasing phosphopantetheinylation of a substrate in a cell, wherein the cell is an E. You have opportunities open to you that you may never have again like high adventure sports, internships, free classes in multiple fields, professional level equipment for student use, and if you're exceptionally lucky, study abroad. As the 1920s began swinging and roaring, more and more universities picked up on the trend of sending students to foreign countries, at least for a time.
For the universities, it can increase the prestige of the institute and allow for more connection to professionals in other countries. They can allow students access to research opportunities not available in their country and let them expand their knowledge of a given field by meeting experts from different countries. Whatever happens, please thank them as best you can and remember to accept the offered help.
Your unofficial tour guide will also probably have a better understanding of the nooks and crannies of the city so pay attention to them.
The enzyme can be purified from a natural source, produced recombinantly, or synthetically.
Strain PCC 7120 Implicates a Secondary Metabolite in the Regulation of Heterocyst Spacing”, Journal of Bacteriology, vol. International influence and recognition may also be achieved with these programs facilitating better interaction between universities in research. This special breed may not necessarily be paid to take people around cities to see famous places. This woman or man is probably going to be just as confused as you are with this place they're in. Professors are an integral part of study abroad as you will be learning from them while you're there.
Okay so technically you're not meeting them, but you're going to need to keep in touch with your family and friends somehow while you're out of the country.
Accordingly, the invention provides compositions and kits including phosphopantetheinyl transferases and host cells expressing phosphopantetheinyl transferases.
These can also pull in more international students as names of foreign universities become more common.
They may speak the same language as you if you're lucky, but they might even speak an entirely separate one, one that you may not know. Taking on the figure of your mother while your real one worries from afar, this woman will help you figure out who will help you and who will hurt you here. The invention also provides nucleic acids encoding phosphopantetheinyl transferases and vectors comprising such nucleic acids.
No matter how good your map is, if you're in a foreign city, you just don't know the lay of the land.
This person will be invaluable to you as you try to go about daily life and school in another country. Just like getting chummy with a professor back home will help you do well in class, it will doubly help if the professor can speak your native language. The invention further provides methods for phosphopantetheinylating a substrate in vitro or in vivo and methods for producing antibiotics in vitro or in vivo. Trans., 21, 202S) and the solution structure of ACP has been solved by NMR spectroscopy (Holak, T.
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. 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 ?-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. 252:39-45), but remarkably little has been shown about the mechanism or specificity of this post-translational phosphopantetheinylation process. 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.BRIEF DESCRIPTION OF THE DRAWINGSFIG.
1 is a schematic representation of the transfer of a 4?-PP moiety from CoA to Ser-36 of apo-ACP by ACPS to produce holo-ACP and 3?,5?-ADP in a Mg2+ dependent reaction.
Holo-ACP can then activate acyl groups for acyl transfer through a thioester linkage to the terminal cysteamine of the 4?-PP moiety.FIG.



2 Panel A which confirms the introduction of [3H]phosphopantetheine into holo-ACP (I), holo-ACP-CoA mixed disulfide (II), and (holo-ACP)2 disulfide (III).FIG.
3 shows the results of Tris-Tricine SDS-PAGE analysis of fractions from purification of wild-type and recombinant ACPS (lane 1, crude lysate of E. 4 is a graphic representation of the number of pmol of holo-Dcp formed per number of pmol of apo-Dcp incorporated in an in vitro phosphopantetheinylation reaction with recombinant E. 5 is a schematic representation of the transfer of an acyl group onto the sulfhydryl group of an ACP by fatty acid synthases (FASs) and polyketide synthases (PKSs) and the transfer of an amino-acyl group onto the sulfhydryl group of an ACP by peptide synthesases.FIG. 7 is a schematic diagram of homologous regions (shaded) among fatty acid synthases (FAS) from S.
9 Panel B shows amino acid sequence alignments of the consensus sequences of the P-pant transferase enzyme superfamily. 11 represents a histogram showing Sfp, EntD, and o195 mediated incorporation of [3H]-4?-Ppant into ACP and His6-PCP.FIG. 13 is a graphical representation of the amount of [3H]-4?-Ppant incorporated into PCP-His6 during a 15 minute reaction with 13 nM Sfp and different concentrations of the substrate.FIG.
14 Panel B is a diagram representing the amount of Holo-ACP formed as a function of time upon incubation of Apo-ACP with EntD, ACPS, or o195.FIG. The term “phosphopantetheinyl transferase” and “phosphopantetheinyl transferase enzyme” and “phosphopantetheinylating enzyme” is intended to include a molecule, e.g. Accordingly, phosphopantetheinyl transferases include natural enzymes, recombinant enzymes, or synthetic enzymes, or active fragments thereof, which are capable or phosphopantetheinylating a compound.
The term phosphopantetheinyl transferase also includes enzymes which modify non-acyl carrier proteins, which require a 4?-phosphopantetheine group, for example, for enzymatic activity.
The terms “isolated” and “purified” are used interchangeably herein and refer to a phosphopantetheinyl transferase or active fragment thereof that is substantially free of other components of the host organism with which it is associated in its natural state.
The terms “isolated” and “purified” are also used to refer to a phosphopantetheinyl transferase or active fragment that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.In one embodiment, the phosphopantetheinyl transferase or active fragment thereof is purified from a cell naturally expressing the synthase, such as a procaryotic or eucaryotic cell. Since ACPs are involved in fatty acid biosynthesis, it is likely that all species having fatty acids would require phosphopantetheinyl transferases for their synthesis. Procaryotic cells which produce phosphopantetheinyl transferase include bacteria, such as E. Phosphopantetheinyl transferases can also be isolated from eucaryotic cells, for example mammalian cells, yeast cells, and insect cells.
Other sources for phosphopantetheinylating enzymes include plant tissues, such as spinach and pea leaves (Elhussein, et al. However, it is preferred that the process of purification of a phosphopantetheinyl transferase from a native source include an affinity purification step over an apo-acyl carrier protein column, such as is further detailed in the Exemplification Section. Use of this affinity purification step allows purification of a phosphopantetheinyl transferase to at least about 60% purity, more preferably to at least about 70% purity, and most preferably to at least about 80% purity.
Purification of a phosphopantetheinyl transferase to at least about 90% purity, 95% purity, 97% purity, 98% purity or 99% purity by this technique is preferred. In addition, purification of a phosphopantetheinyl transferase from a natural source by the techniques described herein will preferably result in an enrichment of the phosphopantetheinyl transferase by at least about 800 fold or 1,000 fold, more preferably at least about 10,000 fold, more preferably at least about 50,000 fold, and even more preferably at least about 70,000 fold. Host cell, expression vectors and techniques for expression of heterologous nucleic acids are known to those skilled in the art.
For example, using recombinant techniques, a phosphopantetheinyl transferase can be expressed as an intracellular protein, a membrane associated protein, or as a secreted protein.
In one embodiment of the invention, a phosphopantetheinyl transferase or active fragment thereof is expressed in a procaryotic cell, such as a bacterial cell, preferably E. Alternatively, the phosphopantetheinyl transferase or fragment thereof can be expressed in a eukaryotic cell, such as a yeast cell, a mammalian cell, such as the CHO or COS cells, or in an insect cell (baculovirus system). The phosphopantetheinyl transferase or fragment thereof can also be expressed in a plant cell. Nucleic acid regulatory elements required for the expression of nucleic acid encoding a phosphopantetheinyl transferase or fragment thereof in the various systems, and methods for introducing one or more nucleic acids in these host cells are well known in the art.
A recombinantly produced phosphopantetheinyl transferase may have an activity identical to that of its natural counterpart, or it may have an activity which varies more or less from that of its natural counterpart. For example, a recombinantly produced phosphopantetheinyl transferase can vary in the efficiency of catalysis. The term “overexpression” of a phosphopantetheinyl transferase is intended to include expression of the nucleic acid encoding a phosphopantetheinyl transferase to levels which are about 100 fold higher, more preferably about 1,000 fold higher, more preferably about 10,000 fold higher, and even more preferably about 100,000 fold higher than the level of the expression of the endogenous phosphopantetheinyl transferase.Various methods can be used for isolating and purifying recombinant phosphopantetheinyl transferase or fragment thereof from a host cell, which are preferably adapted to the specific host system used for expression. A preferred method of purification of a phosphopantetheinyl transferase or fragment thereof comprises an apo-acyl carrier protein affinity purification step. A method of purification of phosphopantetheinyl transferase including such an affinity purification step is described in the Exemplification section.
Purification of a recombinant phosphopantetheinyl transferase or fragment thereof by this technique results in a preparation of phosphopantetheinyl transferase or active fragment thereof having a purity of at least about 90% or greater, preferably at least about 95% purity, more preferably at least about 97% purity, more preferably at least about 98% purity, and even more preferably at least about 99% purity.
An “active fragment” of a phosphopantetheinyl transferase is intended to include a fragment of a phosphopantetheinyl transferase that is capable of phosphopantetheinylating a substrate. Active fragments of phosphopantetheinyl transferases are included in the term phosphopantetheinyl transferases as used herein, since active fragments of phosphopantetheinyl are capable of transfering a phosphopantetheine to a substrate. Active fragments can be produced using the techniques previously described herein for the preparation of purified phosphopantetheinyl transferase, recombinant phosphopantetheinyl transferase, or chemically synthesized using known techniques.
For example, a peptide fragment of a phosphopantetheinyl transferase can be obtained by expressing nucleic acid fragments of a nucleotide sequence encoding a phosphopantetheinyl transferase in an appropriate host cell and screening the resulting peptide fragments for activity using known techniques.
Various methods are known in the art to prepare libraries of clones expressing various fragments of a phosphopantetheinyl transferase. These clones can then be screened to identify those which express an active fragment capable of phosphopantetheinlyating a substrate. In one method, a fragment of a phosphopantetheinyl transferase is tested in an in vitro assay in which apo-acyl carrier protein and CoA having a radiolabelled 4?-phosphopantetheine group is incubated with the test phosphopantetheinyl transferase fragment in an appropriate buffer.
The amount of radiolabel incorporated in the holo-acyl carrier protein is then measured and is representative of the activity of the fragment of the phosphopantetheinyl transferase. Further details regarding this in vitro test are provided in the Exemplification section.Other compounds within the scope of the invention include modified phosphopantetheinyl transferase or modified active fragments thereof. The term “modification” is intended to include addition, deletion, or replacement of one or more amino acid residues in the phosphopantetheinyl transferase or active fragment thereof, such that the phosphopantetheinylating activity of the enzyme is either maintained, increased, or decreased.
For example, a modified phosphopantetheinyl transferase or modified active fragment thereof may be capable of phosphopantetheinylating a different acyl carrier protein than the non-modified phosphopantetheinyl transferase. Alternatively, the modified phosphopantetheinyl transferase may have a different range of specificity. In particular, modification of the enzyme can result in a phosphopantetheinyl transferase capable of phosphopantetheinylating additional substrates.
On the other hand, modification of a phosphopantetheinyl transferase can result in a phosphopantetheinyl transferase with a more restricted range of substrates.
It is possible to determine the target(s) of a phosphopantetheinyl transferase by, for example, performing in vitro phosphopantetheinylation tests, such as described above or in the examples and replacing the E. Other modified phosphopantetheinyl transferases include those wherein a “tag” is attached to the phosphopantetheinyl transferase or active fragment thereof, such as for facilitating purification of the protein from the host cell producing it. The term “homolog” of a phosphopantetheinyl transferase is intended to include phosphopantetheinyl transferases from species other than the E.
It has been shown, for example, that plant tissues have a phosphopantetheinyl transferase, and it is highly likely that most cells, whether eukaryote or prokaryote also contain at least one enzyme which is capable of phosphopantetheinylating substrates. Homologs can also be cloned by other methods known in the art, such as by PCR methods using degenerate oligonucleotides having a nucleic acid sequence derived from that of E.
Homology, also termed herein “identity” refers to sequence similarity between two proteins (peptides) or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.
When a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are homologous, or identical, at that position.
A degree (or percentage) of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
A degree or percentage of “identity” between amino acid sequences refers to amino acid sequence similarity wherein conserved amino acids are considered to be identical for the purpose of determining the degree or percentage of similarity. It has in fact been shown, as described herein, that specific proteins or peptides having limited overall amino acid sequence homology with E. Even though EntD, Sfp, and o195 have only limited overall amino acid sequence homology with E. Further sequence comparison revealed that these regions of conserved amino acid residues are also present in the following proteins: the Fas2 protein from several organisms, including S.
Based at least in part on these amino acid sequence homologies, Fas2, Gsp, and Lpa are also likely to be phosphopantetheinyl transferases. Mutagenesis studies have further provided evidence that these conserved regions are required for phosphopantetheinylation of substrates.Accordingly, preferred phosphopantetheinyl transferases of the invention have at least one or more conserved amino acids, such as the asterisked amino acids shown in FIG.
Yet further preferred phosphopantetheinyl transferases of the invention comprise a motif 1 (a or b) and motif 2 (a or b) separated by at least about 5, preferably at least about 10, more preferably at least about 15, 20, 25, 30, 35, 40, 45, 50, and 55 amino acid residues. In a preferred embodiment, the two motifs are separated by at least about 30 to 45 amino acid residues.
Other preferred phosphopantetheinyl transferases of the invention include those comprising amino acid sequences which are significantly homologous or similar to the amino acid sequence of motifs 1a, 1b, 2a, or 2b.


Also within the scope of the invention are phosphopantetheinyl transferases which contain a motif 1 and a motif 2 which contain amino acid substitutions, deletions, or additions. 9 and a motif 2 having an amino acid sequence listed above or indicated in the right column of FIG. Such assays are preferably preformed on various substrates since certain phosphopantetheine transferases show substrate specificity.Preferred phosphopantetheinylating enzymes have an amino acid sequence identity or similarity of at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, and most preferably at least about 90% with an amino acid sequence of a motif 1 and a motif 2 shown above or in any Figure. Peptides having an amino acid sequence identity or similarity of at least about 93%, more preferably at least about 95%, and most preferably at least about 98-99% with an amino acid sequence of a motif 1 and a motif 2 shown above or in any Figure are also within the scope of the invention.Based on the existence of amino acid sequence homologies between phosphopantetheinylating enzymes, it is possible to identify additional phosphopantetheinylating enzymes. Acyl-CoAs can be used for fatty acid synthesis (FAS) and all the polyketide synthases (PKS), whereas aminoacyl-AMPs can be used for the peptide synthetases (FIG. It has in fact been shown that a phosphopantetheinyl transferase from one species is capable of phosphopantetheinylating a substrate from another species. In yet another embodiment, the phosphopantetheinylating enzyme has a wide range of substrates.This invention further provides kits for in vitro phosphopantetheinylation of substrate proteins. Such kits include an isolated or purified phosphopantetheinyl transferase or active fragment thereof as described herein and instructions for use.
The phosphopantetheinyl transferase or active fragment thereof can be produced by recombinant technique, chemically synthesized, or purified from a native source.
The phosphopantetheinyl transferase or fragment thereof can be packaged, such that it retains substantially all of the phosphopantetheinylation activity for at least one month, preferably for at least 2 months, even more preferably for at least 3 months. Even more preferably, the phosphopantetheinyl transferase is stable for at least 6 months and most preferably for at least 1 year. For example, the phosphopantetheinyl transferase or fragment thereof can be maintained at ?20° C.
Alternatively, the phosphopantetheinyl transferase or fragment thereof can be provided as a lyophilized powder. The kits can further include a 4?-phosphopantetheinylate group providing reagent, such as Coenzyme A (CoA). In addition, an appropriate reaction buffer for the phosphopantetheinylation reaction, preferably in a concentrated form can be included in the kit.
The buffer can be a buffer which maintains or increases the stability of the transferase or which permits the transferase to be maintained in a form under which it maintains its activity or in which its activity can be recovered once the transferase is incubated in appropriate conditions.
For example, the buffer can contain an appropriate amount of glycerol, or any other solution allowing the enzyme to be frozen and to be active upon thawing of the transferase. The phosphopantetheinyl transferase can also be in a lyophilized form.Another embodiment of the invention provides host cells modified to express at least one nucleic acid encoding a phosphopantetheinyl transferase or fragment thereof. Host cells expressing or overexpressing a phosphopantetheinyl transferase have been described herein.
In addition, this invention provides host cells transformed with a nucleic acid encoding a non-secreted form of a phosphopantetheinyl transferase or fragment thereof and at least one additional nucleic acid encoding at least one polypeptide.
In a preferred embodiment, the at least one polypeptide is a component of the substrate of the phosphopantetheinyl transferase. Thus, in one embodiment, the invention provides a method for expressing an active, recombinant form of an enzyme requiring a 4?-phosphopantetheine group by expressing or overexpressing both the enzyme and a phosphopantetheinyl transferase in the same host cell. The enzyme coexpressed in the host cell can be any enzyme which requires phosphopantetheinylation to be active in a reaction. Such compounds include polyketides (both aromatic and macrolide) and non-ribosomally produced peptides, including depsipeptides, lipopeptides, and glcyopeptides.
Biosynthetic pathways that can be modulated with the phosphopantetheinylate transferases of the invention include fatty acids, lipopeptide surfactants and antibiotics. Preferred antibiotics are those non-ribosomally produced peptides, which can be synthesized by a thiotemplate mechanism. Other antibiotics within the scope of the invention include erythromycin, clarythromycin, oxytetracycline, bacitracin, cyclosporin, penicillins, cephalosporins, vancomycin.
Type I ACPS include erythromycin, rapamycin, cyclosporin, avenectin, and tetracycline synthases. The modified host cell is incubated in a medium containing the necessary substrates for the multienzyme polypeptide.
Alternatively, compounds requiring a type I ACP for their synthesis can be produced in vitro with purified enzymes or with recombinantly produced enzymes.Type II ACPS (also termed type II synthase) are discrete proteins which are capable of associating with several other enzymes to form a multienzyme synthase complex. Type II ACPs are components of tetracyclin, gramicidin, tyrocidin, anthrocyclin, and bacitracin synthetases. Thus, this invention provides a method for producing an antibiotic whose synthesis is catalyzed by a type II ACP by introducing into, and expressing in, a host cell the nucleic acids encoding at least some or all the subunits of the multienzyme complex and a nucleic acid encoding a phosphopantetheinyl transferase and incubating such a modified host cell in a medium containing the appropriate substrates for the enzymes. Alternatively, compounds requiring a type II ACP in their synthesis can be produced in vitro, by contacting purified phosphopantetheinyl transferase(s) and the appropriate enzymes necessary for synthesis of the compound, in an appropriate buffer.Preferred host cells for producing antibiotics include cells for which the antibiotic is non toxic. Alternatively, a modified form of the antibiotic that is non toxic to the cell can be expressed and the modified form altered in vitro or in vivo to obtain the active form of the antibiotic.Also within the scope of the invention are organisms which naturally produce antibiotics and which have been modified to express or overexpress a phosphopantetheinyl transferase.
The newly introduced -SH of the phosphopantetheine (P-pant) prosthetic group now acts as a nucleophile for acylation by a specific substrate, i.e. In the PKS complexes the carboxy-activated malonyl-ACP derivative then undergoes decarboxylation, forming a nucleophilic carbanion species which attacks a second acyl thiolester to yield a new carbon-carbon bond polyketide biosynthesis. In peptide and depsipeptide synthetases, the aminoacyl-ACPs or hydroxyacyl-ACPs serve as nucleophiles in amide and ester bond-forming steps respectively. In another embodiment, the invention provides phosphopantetheinyl transferase molecules which have additional enzymatic activities, such as a molecule which has both a phosphopantetheinyl transferase and an ACP activity.
For example, the yeast fatty acid synthase subunit II (FAS II) has been shown herein to have a domain homologous to E.
Accordingly, the invention provides polyfunctional or polyenzymatic proteins comprising as one of the various enzymatic functions, the ability to transfer phosphopantetheinyl groups. Such transfer can be onto the same or another molecule.The invention further provides proteins which do not normally possess the ability to phosphopantetheinylate, but which may be modified to obtain this enzymatic activity. In a preferred embodiment, at least one phosphopantetheinyl transferase or active fragment thereof is linked to a protein which is an ACP. Thus, also within the scope of the invention are methods for producing such compounds using the phosphopantetheinyl transferases, active fragments thereof and expression systems described herein.Methods for producing purified phosphopantetheinyl transferase are also within the scope of the invention.
In one embodiment, a phosphopantetheinyl transferase or active fragment thereof is isolated from a cell naturally producing the enzyme, such as E. Methods for isolating the enzyme preferably include an affinity purification step over a column containing apo-acyl carrier protein.
The invention also provides methods for producing recombinant forms of phosphopantetheinyl transferases. Such methods comprise transforming a host cell with a nucleic acid encoding a phosphopantetheinyl transferase, under conditions appropriate for expression of the protein and isolating the synthase from the host cells and culture media.
An inhibitory compound is defined as a compound that reduces or inhibits phosphopantetheinylation of a substrate by a phosphopantetheinyl transferase.
Inhibitors of phosphopantetheinyl transferase can be identified by using an in vitro or an in vivo test.
In one embodiment, potential inhibitory compounds are screened in an in vitro phosphopantetheinylation assay, such as an assay described in the Exemplification section.
In another embodiment, potential inhibitory compounds are contacted with a microorganism having a phosphopantetheinyl transferase and the phosphopantetheinylation of substrate is monitored. Inhibitory compounds can be used, for example, for blocking specific pathways requiring phosphopantetheinylation in microorganisms, such as ACP dependent pathways.
ACPs are involved, for example, in the transacylation of proteins, such as haemolysin, a pathogenic factor of E. The identification of the yeast protein Lys 2, involved in a primary lysine biosynthetic pathway, as a phosphopantetheinylating enzyme, provides a method for killing yeast. Accordingly, inhibitors of phosphopantetheinylating enzymes can be used as anti-fungal agents.
Moreover, many membrane-associated proteins are acylated and blocking or reducing acylation of these proteins in a microorganism can potentially affect the viability of the microorganism. ACPs have also been involved in transacylation of cell wall components, which are popular therapeutic targets.
Other reactions involving ACPs include transacylation of oligosaccharides, involved for example in the nodulation factors of Rhizobia genus.
Transacylation of oligosaccharides are an essential step in nodule formation in nitrogen fixing plants and has agricultural applications.Yet other embodiments within the scope of the invention include screening methods to uncover novel antibiotics. Similarly, the invention provides methods for isolating novel immunosuppressant, antifungal, antihelminthic, antiparasitic, and antitumor agents.It has been shown herein that the E.
In one embodiment, the invention provides a method for improving iron uptake of bacteria, such as by expressing in bacteria the gene EntD. Improved iron uptake can result in more efficient bacterial growth, which will be useful in cases where bacteria are grown for the production of a beneficial compound.
The invention also provides methods for reducing iron uptake by bacteria, such as by growing bacteria in the presence of an inhibitor of phosphopantetheinylation.



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