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  Methods and compositions for determining enzymatic activityUSPTO Application #: 20070298438Title: Methods and compositions for determining enzymatic activity Abstract: The present invention comprises crystalline polyketide synthases, isolated non-native polyketide synthases having the structural coordinates of said crystalline polyketide synthases, and nucleic acids encoding such non-native polyketide synthases. Also disclosed are methods of predicting the activity and/or substrated specificity of putative polyketide synthase, methods of identifying potential polyetide synthase substrates, and methods of identifying potential polyketide synthase inhibitors. (end of abstract) Agent: Foley & Lardner LLP - San Diego, CA, US Inventors: Joseph P. Noel, Jean-Luc Ferrer, Joseph Jez, Mike Austin, Marianne Bowman USPTO Applicaton #: 20070298438 - Class: 435007400 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, To Identify An Enzyme Or Isoenzyme The Patent Description & Claims data below is from USPTO Patent Application 20070298438. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to methods for designing mutant polyketide synthases, and to predicting the activity and/or substrate specificity of putative native and mutant polyketide synthases. The present invention further relates to methods for identifying polyketide synthase substrates and/or inhibitors. BACKGROUND [0002] Advances in molecular biology have allowed the development of biological agents useful in modulating protein or nucleic acid activity or expression, respectively. Many of these advances are based on identifying the primary sequence of the molecule to be modulated. For example, determining the nucleic acid sequence of DNA or RNA allows the development of antisense or ribozyme molecules. Similarly, identifying the primary sequence allows for the identification of sequences that may be useful in creating monoclonal antibodies. However, often the primary sequence of a protein is insufficient to develop therapeutic or diagnostic molecules due to the secondary, tertiary or quartenary structure of the protein from which the primary sequence is obtained. The process of designing potent and specific inhibitors or activators has improved with the arrival of techniques for determining the three-dimensional structure of an enzyme or polypeptide to be modulated. [0003] The phenylpropanoid synthetic pathway in plants produces a class of compounds know as anthocyanins, which are used for a variety of applications. Anthocyanins are involved in pigmentation and protection against UV photodamage, synthesis of anti-microbial phytoalexins, and are flavonoid inducers of Rhizobium modulation genes 1-4. As medicinal natural products, the phenylpropanoids exhibit cancer chemopreventive activity, as well as anti-mitotic, estrogenic, anti-malarial, anti-oxidant, and antiasthmatic activities. The benefits of consuming red wine, which contains significant amounts of 3,4',5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the dietary importance of these compounds. Chalcone synthase (CHS), a polyketide synthase, plays an essential role in the biosynthesis of plant phenylpropanoids. [0004] An improvement in the understanding of the structure/function of these enzymes would allow for the exploitation of the synthetic capabilities of known enzymes for production of useful new chemical compounds, or allow for the creation of novel non-native enzymes having new synthetic capabilities. A need exists, therefore, for a detailed understanding of the molecular basis of the chemical reactions involved in polyketide synthesis. The present invention addresses this and related needs. SUMMARY OF THE INVENTION [0005] In accordance with the present invention there are presented crystalline polyketide synthases and the three-dimensional coordinates derived therefrom. Three-dimensional coordinates have been obtained for an active form of chalcone synthase and several inactive mutants thereof, both with and without substrate or substrate analog. Similar results have been obtained for the polyketide synthases stilbene synthase and pyrone synthase. [0006] One aspect of the present invention that is made possible by results described herein is that the three-dimensional properties of polyketide synthase proteins are determined, in particular the three-dimensional properties of the active site. The invention features specific coordinates of at least fourteen a carbon atoms defined for the active site in three-dimensional space. R-groups attached to said .alpha.-carbons are defined such that mutants can be made by changing at least one R-group found in the synthase active site. Such mutants may have unique and useful properties. Thus, in another embodiment of the invention, there are provided isolated non-native (e.g., mutant) synthase(s) having at least fourteen active site .alpha.-carbons having the structural coordinates disclosed herein and one or more R-groups other than those found in native chalcone synthase(s). [0007] The three-dimensional coordinates disclosed herein can be employed in a variety of methods. The polyketide synthase used in the crystallization studies disclosed herein is a chalcone synthase derived from Medicago sataiva (alfalfa). A large number of proteins have been isolated and sequenced which have primary amino acid sequence similar to that of chalcone synthase, but for which substrate specificity and/or product is unknown. Thus, in another embodiment of the present invention, there are provided methods for predicting the activity and/or substrate specificity of a putative polyketide synthase. There are further provided methods for identifying potential substrates for a polyketide synthase, as well as inhibitors thereof. [0008] Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification. BRIEF DESCRIPTION OF FIGURES [0009] FIG. 1A presents the chemical structures of chalcone, naringenin, resveratrol, and cerulenin. FIG. 1B presents final SIGMAA-weighted 2Fo-Fc electron density map of the CHS-resveratrol complex in the vicinity of the resveratrol binding site. The map is contoured at 1.sigma.. [0010] FIG. 2A shows a ribbon representation of the CHS homodimer. The approximate alpha carbon positions of Met 137 from each of the monomers are labeled accordingly. Naringenin completely fills the coumaroyl-binding and cyclization pockets while the CoA binding tunnels are highlighted by black arrows. Produced with MOLSCRIPT and rendered with POV-Ray. FIG. 2B presents a stereoview of the monomer's alpha carbon backbone. The orientation of the left-hand monomer is exactly the same as in FIG. 2A. Every twenty residues are numbered starting with residue 3 and include the C-terminal residue, 389. [0011] FIG. 3 shows a comparison of chalcone synthase and 3-ketoacyl-CoA thiolase. Ribbon view of the CHS monomer is oriented perpendicular to the dimer interface. The active site cysteine (Cys 164) and the location of bound CoA are rendered as ball and stick models. In addition, strands .beta.1d and .beta.2d of the cyclization pocket are noted. The reaction catalyzed by CHS is illustrated with the coumaroyl- and malonyl-derived portions of chalcone, respectively. The thiolase monomer is depicted in the same orientation as CHS with the Active site cysteine (Cys 125) modeled and the reaction of thiolase as indicated. Figure prepared with MOLSCRIPT and rendered with POV-Ray. [0012] FIG. 4 collectively shows structures of CHS-Acyl-CoA complexes. The ribbon diagram in panel FIG. 4A (on the top left) is the same as FIG. 2A. The CoA binding region depicted in stereo is bounded by a black box in the upper ribbon diagram. Close-up stereoviews of the C.sub.164S mutant CoA binding region for the malonyl- and hexanoyl-CoA complexes are depicted in FIGS. 4B and 4C, respectively. This mutant retains decarboxylation activity and an acetyl-CoA complex is observed crystallographically for the malonyl-CoA complex. In each complex, placement of the Met 137 loop originating from the dyad-related molecule spatially defines one wall of the cyclization pocket. Hydrogen bonds are depicted as spheres. Figure prepared with MOLSCRIPT and rendered with POV-Ray. [0013] FIG. 5A shows the CHS-naringenin complex viewed down the CoA-binding tunnel. The ribbon diagram at the top left has been rotated 90 degrees around the y-axis from the orientation shown in FIG. 2A. This view approximates the global orientation of the CHS dimer used for the close-up view of the natingenin binding site depicted in stereo. Again, the black box highlights the region of CHS shown in stereo close-up. Hydrogen bonds are depicted as dashed cylinders. FIG. 5B illustrates a comparison of the CHS apoenzyme, CHS-naringenin, and CHS-resveratrol structures. Protein backbone atoms for the three refined structures (apoenzyme, naringenin, and resveratrol) were superimposed by least squares fit in O. The position of bound naringenin and resveratrol are shown. For reference, a modeled low energy conformation of chalcone is indicated by dashed cylinders. Strands .beta.1d and .beta.2d for each complex are also depicted (see FIG. 3). .beta.2d does not change in all the complexes examined, but .beta.1d moves in the CHS-resveratrol complex. FIG. 5C presents representative sequence alignment of the .beta.1d-.beta.2d region is given with positions 255, 266, and 268 highlighted. The first three sequences follow a CHS-like cyclization pathway, while the last three use the STS-cyclization pathway. Figure prepared with MOLSCRIPT and rendered with POV-Ray. [0014] FIG. 6 presents the proposed reaction mechanism for chalcone synthesis. The three boxed regions labeled 1, 2, and 3 depict the addition of acetate units derived from malonyl-CoA during the elongation of polyketide intermediates. Box I is depicted in expanded fashion to illustrate the mechanistic details governing the decarboxylation, enolization, and condensation phase of ketide elongation. Smaller black arrows depict the flow of electrons. Each acetate unit of the malonyl-CoA thioesters is coded to emphasize the portions of chalcone derived from each of three elongation reactions using malonyl-CoA. Cyclization and aromatization of the enzyme bound tetraketide leads to formation of chalcone. Hydrogen bonds are shown as dashed lines. Coenzyme A is symbolized as a circle. [0015] FIG. 7 collectively presents three-dimensional models of the elongation and cyclization reaction in CHS and STS. Views are shown in stereo. FIG. 7A illustrates the elongation of the triketide covalently attached to Cys 164 by the acetyl-CoA carbanion produces the tetraketide CoA thioester reaction intermediate that subsequently reattaches to Cys 164. FIG. 7B illustrates the folding of the tetraketide intermediate in CHS positions the oxygen of C1 near the hydrogen of C6 facilitating internal proton transfer and expulsion of chalcone upon cyclization. FIG. 7C illustrates alternate folding of the tetraketide intermediate and positioning of the oxygen of C7 near the hydrogen of C2 in STS allows formation of resveratrol using an internal proton transfer followed by hydrolysis and decarboxylation. Rendered and dashed lines illustrate potential hydrogen bonding interactions. Figure prepared with MOLSCREPIT and rendered with POV-Ray. [0016] FIG. 8 presents a comparison of the active site volumes of CHS and GCHS2. The active site volumes available for binding ketide intermediates were calculated with VOID00 for the CHS-COA complex and for a homology model of GCHS2 with CoA. The cavities are shown as a wire mesh. The homology model of GCHS2 was generated using MODELER and the volume calculated and displayed as for CHS. The numbering scheme is for alfalfa CHS homodimer. Figure prepared with MOLSCRIPT and rendered with POV-Ray. [0017] FIG. 9 shows an example of a computer system in block diagram form. DETAILED DESCRIPTION OF THE INVENTION [0018] The phenylpropanoid synthetic pathway in plants produces a class of compounds know as anthocyanins, which are used for a variety of applications. Anthocyanins are involved in pigmentation and protection against UV photodamage, synthesis of anti-microbial phytoalexins, and are flavonoid inducers of Rhizobium modulation genes 1-4. As medicinal natural products, the phenylpropanoids exhibit cancer chemopreventive activity, as well as anti-mitotic, estrogenic, anti-malarial, anti-oxidant, and antiasthmatic activities. The benefits of consuming red wine, which contains significant amounts of 3,4',5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the dietary importance of these compounds. [0019] Polyketides are a large class of compounds and include a broad range of antibiotics, immunosuppressants and anticancer agents which together account for sales of over $5 billion per year. Polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (e.g., tetracyclines and erythromycin), anti-cancer agents (e.g., daunomycin), immunosuppressants (e.g., FK506 and rapamycin), and veterinary products (e.g., monensin) and the like. Many polyketides (produced by polyketide synthases) are valuable as therapeutic agents. Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in length and patterns of functionality and cyclization. Continue reading... Full patent description for Methods and compositions for determining enzymatic activity Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods and compositions for determining enzymatic activity patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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