CRYSTAL STRUCTURES OF POLYKETIDE SYNTHASE FOR COMBINATORIAL BIOSYNTHESIS OF ANTI
Shiou-chuan Tsai, Assistant Professor
Stanford University Stanford, Ca 94305
Grant 2P41RR001209-260541 from National Center For Research Resources IRG: ZRG1
Keywords: X ray crystallography, aromatase, biomedical resource, drug discovery /isolation, enzyme structure, polyketide synthase, structural biology, antibiotic, antineoplastic antibiotic, biological product, combinatorial chemistry, bioimaging /biomedical imaging
Project start date: 2005-05-01
Project end date: 2006-02-28
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Shiou-chuan Tsai
THE KETOREDUCTION AND CYCLIZATION OF AROMATIC POLYKETIDE BIOSYNTHESIS
Shiou-chuan Tsai
University Of California Irvine, Irvine, Ca 92697-7600
Grant 3R01GM076330-04S1 from National Institute Of General Medical Sciences
Abstract: This R01 revision application answers RFA NOT-OD-09-058 (Notice Title NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). This is a revision application for the R01 grant GM076330, titled "The Ketoreduction and Cyclization of Aromatic Polyketide Biosynthesis", whose specific aims were 1. Determine the sequence-structure-function relationship of ketoreductase (KR) that leads to its unique reduction specificity. 2. Determine the sequence-structure-function relationship of aromatase/cyclase (ARO/CYC) that leads to its unique cyclization specificity. 3. Determine the importance of protein-protein interactions on the sequence-structure-function relationship between KR and ARO/CYC. We were pleased to report that our progress towards these three aims has exceeded the proposed timelines. In order to further accelerate the research pace towards understanding polyketide ketoreduction and cyclization, we proposed the following three aims that do not overlap, but rather complementary to the parent R01 aims AIM 1. Determine the molecular basis of ketoreduction using chemical probes. AIM 2. Determine the molecular basis of aromatic polyketide cyclization using chemical probes. AIM 3. Solve crystal structures of chemically cross-linked multi-domain PKS complexes. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long-term biomedical relevance is that the polyketide research community can apply the sequence-structure-function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. The revision will accelerate the tempo of scientific research on polyketide synthase (PKS) and allow for job creation and retention for three graduate students and the hiring of one new postdoctoral researcher, and procuring laboratory equipments to accelerate the tempo of the proposed research. This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities
Keywords: (8S-cis)-10-[(3-Amino-2, 3, 6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8-(hydroacetyl)-1-methoxy-5, 12-naphthacenedione; 14-Hydroxydaunomycin; 5, 12-Naphthacenedione, 10-((3-amino-2, 3, 6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-, (8S-cis)-; ARO; ARO1; Active Sites; Adriamycine; Affect; Affinity; Amino Acid Sequence; Anabolism; Androstenedione Aromatase; Anti-Estrogens; Antibiotic Agents; Antibiotic Drugs; Antibiotics; Aromatase; Aromatase Cytochrome P450; Aureolic Acid; Bicyclo Compounds; Binding; Binding (Molecular Function); Biological; Biological Factors; C 1 Esterase; C1 Esterase; C1 s; C1s; CPV1; CYAR; CYP 19; CYP19; CYP19 Protein; CYP19A1; CYP19A1 gene; CYPXIX; Catalysis; Chemicals; Cloning; Communities; Complement 1 Esterase; Complement 1s; Complement component C1s; Complex; Crystallization; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Cyclization; Cytochrome P-450 CYP19; Cytochrome P-450(AROM); Cytochrome P450 19; Cytochrome P450 19A1; Cytochrome P450, Family 19, Subfamily A, Polypeptide 1; Cytochrome P450, Subfamily XIX; Cytochrome P450, Subfamily XIX (Aromatization of Androgens); DNA Sequence; DOX; Data; Dehydratases; Dehydration; Development; Docking; Doxorubicin; Doxorubicina; EC 2.3.1.85; Engineering; Engineerings; Environment; Enzyme Interaction; Enzyme Kinetics; Enzymes; Equipment; Estrogen Antagonists; Estrogen Synthase; Estrogen Synthetase; Factor, Biologic; Family; Fatty Acid Synthetase Complex; Fatty Acids; Fatty-acid synthase; Figs; Figs - dietary; Funding; Gene Cluster; Genes; Genetic Alteration; Genetic Change; Genetic Engineering of Proteins; Genetic analyses; Genetic defect; Genetics-Mutagenesis; Grant; Hydrases; Hydro-Lyases; Hydroxyl Daunorubicin; Hydroxyldaunorubicin; In Vitro; Individual; Investigation; Investigators; Jobs; Kinetic; Kinetics; Knowledge; Label; Laboratories; Length; Libraries; Ligands; Link; Macrolide Antibiotics; Miscellaneous Antibiotic; Mithramycin; Mitramycin; Modification; Molecular; Molecular Biology, Mutagenesis; Molecular Biology, Protein Sequencing; Molecular Interaction; Multienzyme Complexes; Mutagenesis; Mutate; Mutation; NIH; National Institutes of Health; National Institutes of Health (U.S.); Natural Products; Occupations; Outcome; P-450AROM; P450AROM; PKS enzyme; Parents; Pattern; Peptide Sequence Determination; Pharmaceutical Agent; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Plicamycin; Population; Position; Positioning Attribute; Principal Investigator; Professional Postions; Protein Engineering; Protein Sequencing; Protein Structure, Primary; Proteins; Public Health; Recovery; Reporting; Research; Research Personnel; Researchers; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Sequence Homology; Simulate; Single Crystal Diffraction; Specificity; Step-Parent; Stepparent; Streptomyces; Structure; Structure-Activity Relationship; Substrate Specificity; System; System, LOINC Axis 4; TXT; Testing; Tetracycline Antibiotic; Tetracyclines; Text; TimeLine; Training; Translating; Translatings; Tyr-Lys; United States National Institutes of Health; Variant; Variation; X Ray Crystallographies; X-Ray Crystallography; actinorhodin; antiestrogen; antiestrogenic; antitumor agent; base; bicyclic compound; biosynthesis; body water dehydration; chemical structure function; combinatorial; cross-link; crosslink; enzyme activity; enzyme complex; estrogen inhibitor; gene product; genetic analysis; genome mutation; graduate student; homology (molecular); in vitro Assay; in vivo; innovate; innovation; innovative; language translation; molecular assembly; molecular assembly/self assembly; molecular self assembly; mutant; novel; polyketide synthase; protein complex; protein protein interaction; protein sequence; public health medicine (field); public health relevance; skills; small molecule; stereochemistry; structure function relationship; sugar; tool; tyrosyl-lysine; tyrosyllysine
Relevance: This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities
Project start date: 2010-09-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
3R01GM076330-04S1 (2010): $126345
THE KETOREDUCTION AND CYCILIZATION OF AROMATIC POLYKETIDE BIOSYNTHESIS
Shiou-chuan Tsai, Associate Professor
University Of California Irvine, Irvine, Ca 92697-7600
Grant 5R01GM076330-04 from National Institute Of General Medical Sciences
Abstract: The objective of this proposal is to determine sequence-structure-function relationships of a protein complex called polyketide synthase (PKS), an enzyme complex comprised of 5 - 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. The focus of this proposal is to determine the crystal structures and sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. The central hypothesis ´or the proposed research is that we can use structure-directed mutagenesis to change the substrate specificity and enzyme activity in a predictable manner. We have formulated this hypothesis based on our preliminary results of 14 crystal structures. 30 mutants, five in vitro substrates and kinetic studies of the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO). We will pursue the following specific aims AIM 1. Determine the Sequence-Structure-Function Relationship of Ketoreductase (KR) That Leads to its Unique Reduction Specificity, in which we will (1) determine the cocrystal structures of actKR and substrates/inhibitors in order to identify structural features important for catalysis and protein-ligand nteractions, (2) probe the active site geometry of actKR with polycarbonyl compounds as the "molecular ruler" in order to correlate different ligand binding motifs with active site geometry, and (3) identify residues important for enzyme activity, stereo-specificity and regio-specificity by kinetic assays and structure-directed mutagenesis. AIM 2. Determine the Sequence-Structure-Function Relationship of Aromatase/Cyclase (ARO/CYC) That Leads to its Unique Cyclization Specificity, in which we will (1) determine the co-crystal structures of tcmARO and inhibitors in order to dissect the protein-ligand interactions, (2) determine the crystal structures of different ARO/CYCs in order to identify important sequence-structure features for different biological functions, and (3) identify tcmARO residues important for catalysis and cyclization specificity by kinetic assays and structure-directed mutagenesis; AIM 3. Determine the Importance of Protein-Protein Interactions on the Sequence-Structure-Function Relationship between KR and ARO/CYC, in which we will correlate protein-protein interactions between PKS domains with enzyme activity and regio-specificity. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long- term biomedical relevance is that the polyketide research community can apply the sequence-structure- function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities
Keywords: ARO; ARO1; Active Sites; Affect; Amino Acid Sequence; Anabolism; Androstenedione Aromatase; Aromatase; Aromatase Cytochrome P450; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological; Biological Assay; Biological Factors; Biological Function; Biological Process; C 1 Esterase; C1 Esterase; C1 s; C1s; CPV1; CYAR; CYP 19; CYP19; CYP19 Protein; CYP19A1; CYP19A1 gene; CYPXIX; Catalysis; Cloning; Communities; Complement 1 Esterase; Complement 1s; Complement component C1s; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Cyclization; Cytochrome P-450 CYP19; Cytochrome P-450(AROM); Cytochrome P450 19; Cytochrome P450 19A1; Cytochrome P450, Family 19, Subfamily A, Polypeptide 1; Cytochrome P450, Subfamily XIX; Cytochrome P450, Subfamily XIX (Aromatization of Androgens); Data; Dehydratases; Development; Docking; Environment; Enzyme Interaction; Enzyme Kinetics; Enzymes; Estrogen Synthase; Estrogen Synthetase; Factor, Biologic; Fatty Acids; Figs; Figs - dietary; Genes; Genetic Engineering of Proteins; Genetic analyses; Genetics-Mutagenesis; Hydrases; Hydro-Lyases; In Vitro; Individual; Investigation; Kinetic; Kinetics; Knowledge; Label; Length; Libraries; Ligand Binding; Ligands; Modification; Molecular; Molecular Biology, Mutagenesis; Molecular Biology, Protein Sequencing; Molecular Interaction; Multienzyme Complexes; Mutagenesis; Natural Products; Outcome; P-450AROM; P450AROM; PKS enzyme; Pattern; Peptide Sequence Determination; Pharmaceutical Agent; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Population; Position; Positioning Attribute; Principal Investigator; Protein Engineering; Protein Sequencing; Protein Structure, Primary; Proteins; Public Health; Research; Role; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Simulate; Single Crystal Diffraction; Sites, Active; Specificity; Streptomyces; Structure; Structure-Activity Relationship; Substrate Specificity; Training; Variant; Variation; X Ray Crystallographies; X-Ray Crystallography; actinorhodin; base; biosynthesis; chemical structure function; combinatorial; enzyme activity; enzyme complex; experiment; experimental research; experimental study; expression cloning; gene product; genetic analysis; graduate student; in vitro Assay; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; mutant; novel; polyketide synthase; protein complex; protein protein interaction; protein sequence; public health medicine (field); research study; social role; stereochemistry; structure function relationship; sugar
Project start date: 2007-07-01
Project end date: 2012-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
5R01GM076330-04 (2010): $242130
THE KETOREDUCTION AND CYCLIZATION OF AROMATIC POLYKETIDE BIOSYNTHESIS
Shiou-chuan Tsai, Associate Professor
University Of California Irvine, Irvine, Ca 92697-7600
Grant 5R01GM076330-03 from National Institute Of General Medical Sciences
Abstract: The objective of this proposal is to determine sequence-structure-function relationships of a protein complex called polyketide synthase (PKS), an enzyme complex comprised of 5 - 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. The focus of this proposal is to determine the crystal structures and sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. The central hypothesis ´or the proposed research is that we can use structure-directed mutagenesis to change the substrate specificity and enzyme activity in a predictable manner. We have formulated this hypothesis based on our preliminary results of 14 crystal structures. 30 mutants, five in vitro substrates and kinetic studies of the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO). We will pursue the following specific aims AIM 1. Determine the Sequence-Structure-Function Relationship of Ketoreductase (KR) That Leads to its Unique Reduction Specificity, in which we will (1) determine the cocrystal structures of actKR and substrates/inhibitors in order to identify structural features important for catalysis and protein-ligand nteractions, (2) probe the active site geometry of actKR with polycarbonyl compounds as the "molecular ruler" in order to correlate different ligand binding motifs with active site geometry, and (3) identify residues important for enzyme activity, stereo-specificity and regio-specificity by kinetic assays and structure-directed mutagenesis. AIM 2. Determine the Sequence-Structure-Function Relationship of Aromatase/Cyclase (ARO/CYC) That Leads to its Unique Cyclization Specificity, in which we will (1) determine the co-crystal structures of tcmARO and inhibitors in order to dissect the protein-ligand interactions, (2) determine the crystal structures of different ARO/CYCs in order to identify important sequence-structure features for different biological functions, and (3) identify tcmARO residues important for catalysis and cyclization specificity by kinetic assays and structure-directed mutagenesis; AIM 3. Determine the Importance of Protein-Protein Interactions on the Sequence-Structure-Function Relationship between KR and ARO/CYC, in which we will correlate protein-protein interactions between PKS domains with enzyme activity and regio-specificity. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long- term biomedical relevance is that the polyketide research community can apply the sequence-structure- function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities
Keywords: ARO; ARO1; Active Sites; Affect; Amino Acid Sequence; Anabolism; Androstenedione Aromatase; Aromatase; Aromatase Cytochrome P450; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological; Biological Assay; Biological Factors; Biological Function; Biological Process; CPV1; CYAR; CYP 19; CYP19; CYP19 Protein; CYP19A1; CYP19A1 gene; CYPXIX; Catalysis; Cloning; Communities; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Cyclization; Cytochrome P-450 CYP19; Cytochrome P-450(AROM); Cytochrome P450 19; Cytochrome P450 19A1; Cytochrome P450, Family 19, Subfamily A, Polypeptide 1; Cytochrome P450, Subfamily XIX; Cytochrome P450, Subfamily XIX (Aromatization of Androgens); Data; Dehydratases; Development; Docking; Environment; Enzyme Interaction; Enzyme Kinetics; Enzymes; Estrogen Synthase; Estrogen Synthetase; Factor, Biologic; Fatty Acids; Figs; Figs - dietary; Genes; Genetic Engineering of Proteins; Genetic analyses; Genetics-Mutagenesis; Hydrases; Hydro-Lyases; In Vitro; Individual; Investigation; Kinetic; Kinetics; Knowledge; Length; Libraries; Ligand Binding; Ligands; Modification; Molecular; Molecular Biology, Mutagenesis; Molecular Biology, Protein Sequencing; Molecular Interaction; Multienzyme Complexes; Mutagenesis; Natural Products; Outcome; P-450AROM; P450AROM; PKS enzyme; Pattern; Peptide Sequence Determination; Pharmaceutical Agent; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Population; Position; Positioning Attribute; Principal Investigator; Protein Engineering; Protein Sequencing; Protein Structure, Primary; Proteins; Public Health; Research; Role; Sequence Determinations, Amino Acid; Sequence Determinations, Protein; Simulate; Single Crystal Diffraction; Sites, Active; Specificity; Streptomyces; Structure; Structure-Activity Relationship; Substrate Specificity; Training; Variant; Variation; X Ray Crystallographies; X-Ray Crystallography; actinorhodin; base; biosynthesis; chemical structure function; combinatorial; enzyme activity; enzyme complex; experiment; experimental research; experimental study; expression cloning; gene product; genetic analysis; graduate student; in vitro Assay; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; mutant; novel; polyketide synthase; protein complex; protein protein interaction; protein sequence; public health medicine (field); research study; social role; structure function relationship
Project start date: 2007-07-01
Project end date: 2012-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
5R01GM076330-03 (2009): $246440
The Ketoreduction And Cycilization Of Aromatic Polyketide Biosynthesis
Shiou-chuan Tsai, Assistant Professor
University Of California Irvine Irvine, Ca 926977600
Grant 1R01GM076330-01A2 from National Institute Of General Medical Sciences IRG: MSFB
Abstract: The objective of this proposal is to determine sequence-structure-function relationships of a protein complex called polyketide synthase (PKS), an enzyme complex comprised of 5 - 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. The focus of this proposal is to determine the crystal structures and sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. The central hypothesis or the proposed research is that we can use structure-directed mutagenesis to change the substrate specificity and enzyme activity in a predictable manner. We have formulated this hypothesis based on our preliminary results of 14 crystal structures. 30 mutants, five in vitro substrates and kinetic studies of the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO). We will pursue the following specific aims AIM 1. Determine the Sequence-Structure-Function Relationship of Ketoreductase (KR) That Leads to its Unique Reduction Specificity, in which we will (1) determine the cocrystal structures of actKR and substrates/inhibitors in order to identify structural features important for catalysis and protein-ligand nteractions, (2) probe the active site geometry of actKR with polycarbonyl compounds as the "molecular ruler" in order to correlate different ligand binding motifs with active site geometry, and (3) identify residues important for enzyme activity, stereo-specificity and regio-specificity by kinetic assays and structure-directed mutagenesis. AIM 2. Determine the Sequence-Structure-Function Relationship of Aromatase/Cyclase (ARO/CYC) That Leads to its Unique Cyclization Specificity, in which we will (1) determine the co-crystal structures of tcmARO and inhibitors in order to dissect the protein-ligand interactions, (2) determine the crystal structures of different ARO/CYCs in order to identify important sequence-structure features for different biological functions, and (3) identify tcmARO residues important for catalysis and cyclization specificity by kinetic assays and structure-directed mutagenesis; AIM 3. Determine the Importance of Protein-Protein Interactions on the Sequence-Structure-Function Relationship between KR and ARO/CYC, in which we will correlate protein-protein interactions between PKS domains with enzyme activity and regio-specificity. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long- term biomedical relevance is that the polyketide research community can apply the sequence-structure- function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities.
Keywords: biosynthesis, cyclization, Streptomyces, X ray, X ray crystallography, active site, aromatase, base, biological product, catalyst, community, emotion, environment, enzyme, enzyme activity, enzyme complex, expression cloning, fatty acid, gene, genetics, hydro lyase, lead, library, ligand, mutant, polyketide synthase, protein, protein engineering, protein protein interaction, public health, quality of life, reduction, role, stereochemistry, training
Project start date: 2007-07-01
Project end date: 2012-04-30
1R01GM076330-01A2 (2007): $242018
STRUCTURE-BASED TUBERCULOSIS DRUG DESIGN TARGETED AT ACYL-COA CARBOXYLASE
Shiou-chuan Tsai, Associate Professor
University Of California Irvine, Irvine, Ca 92697-7600
Grant 5R01AI076460-02 from National Institute Of Allergy And Infectious Diseases
Abstract: Principal Investigator/Program Director (Last, First, Middle) Tsai, Shiou-Chuan See instructions. State the application´s broad, long-term objectives and specific aims, making reference to the health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. Mycobacterium tuberculosis, the pathogen of tuberculosis (TB), has a cell envelope with chemically complex lipids that are closely related with its virulence and multi-drug resistance. Acyl-CoA carboxylase (ACCase) provides the building blocks for these complex lipids, and the importance and validity of ACCase as a drug target is well recognized. The M. tuberculosis ACCase include six ACCase subunits (accD1-6), and that AccD4, AccD5 and AccD6 play major roles in providing the building-blocks to cell wall lipid biosyntheses. However, very little is known about the substrate specificity or biological functions of these pathogen ACCases. Our long-term goal is to discover a library of novel anti-TB therapeutics against new M. tuberculosis protein targets. The objective of this particular application is to elucidate the substrate specificities, sequence-structure-function relationship, and biological roles of AccD4, AccD5 and AccD6, using X-ray crystallography, enzyme inhibition assays, and computer-assisted inhibitor design. The rationale is that, once we identified inhibitors of AccD4-6, we will be able to inhibit cell wall lipid biosynthesis, leading to pathogen death. This rationale has been validated by past genetic data, which indicate that mutations of AccD4 and AccD6 lead to pathogen death. In the next two years, we will persue three aims AIM 1. Determine the molecular basis of substrate specificities in AccD4-6 (1.1) Refine the co-crystal structures of AccD5 bound with propionyl-CoA and biotin analogs. (1.2) Refine the co-crystal structures of AccD6 bound with acetyl-CoA and biotin analogs. (1.3) Solve the crystal structure of apo AccD4, and cocrystal structures of AccD4 bound with long chain acyl-CoA and biotin analogs. AIM 2. Determine the inhibitor-binding specificities of AccD5-6 (2.1). Screen in silico Sulfa, Propeller and andrimid (three identified inhibitors) analogs against AccD5-6 using UC Irvine´s ChemDB and cross-validation with two docking softwares. (2.2) Screen in vitro the inhibitors predicted from 2.1 and elucidate the AccD5-6 enzyme mechanisms by inhibition kinetics. (2.3) Refine co-crystal structures of AccD5-6 bound with Sulfa, Propeller or andrimid. AIM 3. Compare the active site geometries and substrate binding pockets of AccD4, AccD5 and AccD6, and define the substrate/inhibitor binding residues by site-directed mutagenesis (3.1) Systematically mutate AccD5 residue 437 to evaluate its importance for substrate specificity. (3.2) Mutate residues in the Acyl-CoA binding pocket to probe for AccD5 specificities for acyl-CoA and Sulfa analogs. The feasibility of the proposed studies are strongly supported by strong preliminary data, including diffracting crystals of all proposed structural studies (AIM 1 and AIM 2.3), as well as established enzyme assays, identification of more than 50 potent inhibitors in AIM 2, and complete construction of half mutants proposed in AIM 3. The proposed research is scientifically significant because, for the first time, the substrate/inhibitor specificities of these unique M. tuberculosis ACCases will be critically evaluated and dissected. Such findings are original, because no ACCase from any other organisms has such a uniquely diverse, yet precisely controlled substrate specificity. The outcome from this proposal will identify potent ACCase inhibitors. Therefore, the completion of this project will also have health significance on the development of new TB therapeutics. The proposed research will retain and increase job opportunities for two graduate students and two postdoctoral researcher, and the outcome will enable us to provide new building blocks for downstream polyketide biosynthesis in an one-pot, environmentally friendly fashion that completes multi-step total syntheses by turning the bacteria into drug-manufacturing factory. PERFORMANCE SITE(S) (organization, city, state) University of California, Irvine, CA 92697, USA REVISED SECTION
Keywords: 1H-Thieno(3, 4-d)imidazole-4-pentanoic acid, hexahydro-2-oxo-, (3aS-(3aalpha, 4beta, 6aalpha))-; 4-Pyridinecarboxylic acid, hydrazide; AIDS; Acetyl CoA; Acetyl Coenzyme A; Acids; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Actinobacteria; Actinobacteria class; Actinomyces; Actinomycetes; Active Sites; Acyl CoA; Acyl Coenzyme A; Affinity; Anabolism; Animals; Anti Mycobacterial Agents; Antimycobacterial Agents; Antitubercular Agents; Antitubercular Drugs; Assay; Bacteria; Binding; Binding (Molecular Function); Bioassay; Biochemical Reaction; Biochemistry; Biologic Assays; Biological; Biological Assay; Biological Factors; Biological Function; Biological Process; Biotin; California; Cell Wall; Cells; Cessation of life; Chemistry, Biological; Cities; Coenzyme A, S-acetate; Commit; Communicable Diseases; Complex; Computer Assisted; Computer Simulation; Computerized Models; Confidential Information; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Data; Death; Development; Docking; Drug Delivery; Drug Delivery Systems; Drug Design; Drug Resistance, Multiple; Drug Resistant, Multiple; Drug Targeting; Drug Targetings; Drug resistance; Drugs; Environment; Enzymatic Reaction; Enzyme Inhibition; Enzymes; Factor, Biologic; Fatty Acids; Fatty Acyl CoA; Funding; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genome; Genus Mycobacterium; Goals; Gram-Positive Bacteria, High G+C; Health; IC50; Immunologic Deficiency Syndrome, Acquired; In Vitro; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Inhibitory Concentration 50; Inorganic Sulfates; Instruction; Investigation; Investigators; Isonicotinic Acid Hydrazide; Jobs; Kinetic; Kinetics; Language; Lead; Libraries; Lipids; Long-Chain Acyl CoA; M. tb; M. tuberculosis; M.tb; M.tuberculosis; Mathematical Model Simulation; Mathematical Models and Simulations; Medication; Methods; Methods and Techniques; Methods, Other; Mission; Models, Computer; Molecular; Molecular Interaction; Multi-Drug Resistance; Multidrug Resistance; Mutagenesis, Site-Directed; Mutate; Mutation; Mycobacterium; Mycobacterium tuberculosis; Mycolic Acid; Natural Products; Occupations; Organism; Outcome; Pb element; Pharmaceutic Preparations; Pharmaceutical Preparations; Plants; Plants, General; Play; Position; Positioning Attribute; Principal Investigator; Professional Postions; Programs (PT); Programs [Publication Type]; Proteins; Public Health; Regulation; Research; Research Design; Research Personnel; Researchers; Resistance to Multi-drug; Resistance to Multidrug; Resistance to Multiple Drug; Resistant to Multiple Drug; Resistant to multi-drug; Resistant to multidrug; Role; SEQ-AN; Sequence Analyses; Sequence Analysis; Simulation, Computer based; Single Crystal Diffraction; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Specificity; Structure; Structure-Activity Relationship; Study Type; Substrate Specificity; Sulfates; Sulfates, Inorganic; Sulfates, Unspecified or Sulfate Ion; Targeted DNA Modification; Targeted Modification; Techniques; Therapeutic; Time; Toxic effect; Toxicities; Training; Tuberculosis; Tuberculostatic Agents; Universities; Unspecified or Sulfate Ion Sulfates; Validation; Virulence; Vitamin H; Work; X Ray Crystallographies; X-Ray Crystallography; ing; adipogenesis; analog; andrimid; anti-microbial; anti-tuberculosis; antimicrobial; antimycobacterial; antituberculosis; assay development; base; biosynthesis; cell envelope; chemical structure function; chemotherapy; coenzyme R; computational modeling; computational models; computational simulation; computer aided; computer based models; computerized modeling; computerized simulation; design; designing; disseminated TB; disseminated tuberculosis; drug resistant; drug/agent; enzyme mechanism; fatty acid biosynthesis; gene product; genome mutation; graduate student; heavy metal Pb; heavy metal lead; improved; in silico; in vivo; inhibitor; inhibitor/antagonist; isoniazid; lipid biosynthesis; lipogenesis; living system; multi-drug resistant; multidrug resistant; mutant; novel; pathogen; performance site; programs; propionyl-CoA; propionyl-coenzyme A; public health medicine (field); resistance to Drug; resistant strain; resistant to Drug; social role; structure function relationship; study design; sulfate; therapeutic target; tuberculosis drugs; tuberculosis treatment; tuberculous spondyloarthropathy; virtual simulation
Project start date: 2009-07-17
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01AI076460-02 (2010): $306000
1R01AI076460-01 (2009): $333650
DISSECTING THE SUBSTRATE SPECIFICITY OF ACYL-COA CARBOXYLASE
Shiou-chuan Tsai, Associate Professor
University Of California Irvine, Irvine, Ca 92697-7600
Grant 1R03AI073426-01A1 from National Institute Of Allergy And Infectious Diseases
Abstract: Acyl-coenzyme A carboxylases (ACCases), such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC), catalyze the carboxylation of acetyl- and propionyl-CoA to provide malonyl- and methylmalonyl-CoA, respectively. This carboxylation reaction is ubiquitously important in biological systems, because it commits acetyl-CoA and propionyl-CoA to the biosyntheses of fatty acids, polyketides and Kreb cycle intermediates. While there is a well-developed body of knowledge on the genetic analysis, mechanistic and biomimetic studies of ACCases, the development of ACCase-related therapeutics has been severely hampered by the lack of molecular information on how ACCases recognize their corresponding substrates or inhibitors. Our long-term goal is to generate ACCase-based therapeutics and to screen for their pharmaceutical activities. The objective of this particular application, which is the next step toward our long-term goal, is to determine the molecular basis of substrate specificity of ACC and PCC from Streptomyces coelicolor. The S. coelicolor ACCases provide extender units to the biosynthesis of polyketides, a class of natural products that include many antibiotic, anticancer and cholesterol-lowering pharmaceuticals. Mutant ACCases can potentially provide new building blocks to polyketide biosynthesis, so that new polyketides with altered extender units can be biosynthesized. These new polyketides, with the antibiotic chemical templates, will be excellent drug leads to be screened against bioterrorism targets of bacteria and viruses. The central hypothesis is that it should be possible to use mutagenesis to change the substrate specificity of ACCase for the purpose of generating new extender units for polyketide biosynthesis. We base the hypothesis on the observation that 1) ACCase subunits have distinct specificity for different substrate and inhibitors; 2) our preliminary data on the structures and functions of the ACCase 2-subunits (AccB and PccB) have identified specific residues that are responsible for molecular recognition. If the hypothesis is true, mutant ACCases will produce new substituted malonyl-CoAs, which can serve as new extender units for polyketide biosynthesis. We will pursue two specific aims AIM 1. SOLVE COCRYSTAL STRUCTURES OF ACCB AND PCCB 1.1. Solve protein-substrate cocrystal structures. 1.2. Solve protein-regulator cocrystal structures. AIM 2. MAKE ACTIVE SITE MUTANTS OF ACCB AND PCCB 2.1. Systematically mutate residue 422. 2.2. Mutate residues in the acyl-CoA binding pocket. 2.3. Mutate residues in the biotin binding pocket. Once we identify the residues that can be mutated to change the specificity of ACCase, it will become possible to generate new, substituted malonyl-CoAs that can serve as new extender units for polyketide biosynthesis. This innovative approach has not been undertaken before. Because of our research focus and the complementary expertise, our research environment is especially conductive to successful completion of the proposed investigations on ACCases. The research proposed in this application is significant, because its outcome allows us to dissect the molecular features that are responsible for substrate specificity of ACCases. In the long run, the result from this proposal will have a significant positive impact on the development of new antibiotics that are either ACCase inhibitors (for blocking fatty acid biosynthesis of bacteria) or have new extender units (for the biosynthesis of new polyketides). Finally, the molecular basis of substrate specificity, determined from the proposed research, will mark a breakthrough in the research of acyl-CoA carboxylase. This project will result in the production of new polyketides that are synthesized with new building blocks. Because polyketides contain many antibiotic and anticancer compounds, the outcome of this project will benefit the general public health by providing new "unnatural" natural products for new drug leads
Keywords: 1H-Thieno(3, 4-d)imidazole-4-pentanoic acid, hexahydro-2-oxo-, (3aS-(3aalpha, 4beta, 6aalpha))-; Acetyl CoA; Acetyl Coenzyme A; Acetyl Coenzyme A Carboxylase; Acetyl-CoA Carboxylase; Acetyl-CoA[{..}]carbon-dioxide ligase (ADP-forming); Active Sites; Acyl CoA; Acyl Coenzyme A; Affect; Anabolism; Antibiotic Agents; Antibiotic Drugs; Antibiotics; Bacteria; Binding; Binding (Molecular Function); Biochemistry; Biological Factors; Biological Terrorism; Biomimetics; Bioterrorism; Biotin; Chemicals; Chemistry, Biological; Cholest-5-en-3-ol (3beta)-; Cholesterol; Coenzyme A, S-acetate; Commit; Communities; Data; Deficiency Diseases; Development; Drugs; Environment; Factor, Biologic; Fatty Acyl CoA; General Population; General Public; Genetic Alteration; Genetic Change; Genetic analyses; Genetic defect; Genetics-Mutagenesis; Goals; Herbicides; Human; Human, General; Investigation; Knowledge; Link; Long-Chain Acyl CoA; Man (Taxonomy); Man, Modern; Medication; Metabolic; Metabolic Diseases; Metabolic Disorder; Mimetics, Biological; Miscellaneous Antibiotic; Molecular; Molecular Biology, Mutagenesis; Molecular Interaction; Multienzyme Complexes; Mutagenesis; Mutate; Mutation; Natural Products; Outcome; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Preparations; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Production; Propionyl-CoA Carboxylase; Propionyl-Coenzyme A Carboxylase; Proteins; Public Health; Reaction; Research; Running; Screening procedure; Sites, Active; Specificity; Streptomyces; Streptomyces coelicolor; Structure; Substrate Specificity; Testing; Therapeutic; Thesaurismosis; Time; Training; Virus; Viruses, General; Vitamin H; anticancer activity; base; biological systems; biosynthesis; carboxylation; coenzyme R; design; designing; drug/agent; enzyme complex; fatty acid biosynthesis; gene product; genetic analysis; genome mutation; graduate student; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; ketotic glycinemia; ketotic hyperglycinemia; metabolism disorder; methylmalonyl CoA; methylmalonyl-CoA decarboxylase; methylmalonyl-coenzyme A; molecular recognition; mutant; new therapeutics; next generation therapeutics; novel therapeutics; propionic acidemia; propionyl coA carboxylase deficiency; propionyl-CoA; propionyl-coenzyme A; public health medicine (field); public health relevance; screening; screenings; structural biology
Relevance: This project will result in the production of new polyketides that are synthesized with new building blocks. Because polyketides contain many antibiotic and anticancer compounds, the outcome of this project will benefit the general public health by providing new "unnatural" natural products for new drug leads
Project start date: 2010-05-01
Project end date: 2012-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PA-06-180
1R03AI073426-01A1 (2010): $69696