Enzymatic formation of the hydrocarbon skeletons underlying diterpenoid diversity

Summary

Principal Investigator: REUBEN JOHN PETERS
Abstract: DESCRIPTION (provided by applicant): Natural products have a proven record of providing a significant fraction, either directly or as lead compounds, of human medicines. Among natural products, the terpenoids (isoprenoids) stand out as being the largest class (>50,000 already known), with the 20-carbon diterpenoids targeted here forming a significant fraction of these (>12,000 known). Indeed, the extensive diversification of diterpenoids indicates that the manifold structures that can be formed from this C20 backbone provide a rich source of biological activity, and a number of these natural products are used as pharmaceuticals (e.g., the anti- cancer paclitaxel/TaxolTM and anti-biotic mutilins) or research tools (e.g., the protein kinase C activating and tumor promoting phorbol esters, and adenylate cyclase activator forskolin). Conversely, we have recently discovered that the human pathogen Mycobacterium tuberculosis utilizes a diterpenoid, isotuberculosinol, to modulate our initial immune response to infection. Accordingly, we propose here to continue our productive investigations of the enzymes, from both plants and microbes, that catalyze formation of the underlying hydrocarbon skeletal structures. Specifically, we will build on our findings from the previous grant period, which includes not only the discovery of isotuberculosinol, but elucidation of some of the first diterpene synthase crystal structures as well. This proposal then advances our long- term goal of engineering enzymes and metabolic pathways for the production of targeted libraries and specific individual terpenoid 'natural'products for pharmaceutical investigation and use. In particular, the objective of this proposal is a detailed structure-function investigation of the synthases/cyclases that catalyze formation of the hydrocarbon skeletons underlying the structural diversity of diterpenoid natural products. These enzymes catalyze complex electrophilic reactions that form new carbon-carbon bonds, and play important roles in initiating diterpenoid biosynthesis. Accordingly, our studies have implications not only for providing increased access to such natural products, but also insights into potential drug targets, specifically in the widespread pathogen M. tuberculosis.
Funding Period: 2005-12-01 - 2015-07-31
more information: NIH RePORT

Top Publications

  1. pmc Synergistic substrate inhibition of ent-copalyl diphosphate synthase: a potential feed-forward inhibition mechanism limiting gibberellin metabolism
    Sladjana Prisic
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Plant Physiol 144:445-54. 2007
  2. pmc Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase
    Mustafa Köksal
    Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA
    Nat Chem Biol 7:431-3. 2011
  3. pmc Domain loss has independently occurred multiple times in plant terpene synthase evolution
    Matthew L Hillwig
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Plant J 68:1051-60. 2011
  4. pmc Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis
    Ke Zhou
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
    J Biol Chem 287:6840-50. 2012
  5. pmc Effect of isotopically sensitive branching on product distribution for pentalenene synthase: support for a mechanism predicted by quantum chemistry
    Liansuo Zu
    Department of Chemistry, University of California, Davis, California 95616, USA
    J Am Chem Soc 134:11369-71. 2012
  6. pmc Terpenoid synthase structures: a so far incomplete view of complex catalysis
    Yang Gao
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Nat Prod Rep 29:1153-75. 2012
  7. pmc Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals
    Yisheng Wu
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Phytochemistry 84:40-6. 2012
  8. pmc Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals
    Ke Zhou
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Phytochemistry 84:47-55. 2012
  9. pmc Functional characterization and evolution of the isotuberculosinol operon in Mycobacterium tuberculosis and related Mycobacteria
    Francis M Mann
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University Ames, IA, USA
    Front Microbiol 3:368. 2012
  10. pmc A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase
    Jared Criswell
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
    Org Lett 14:5828-31. 2012

Research Grants

  1. Synthesis of Complex Terpenes
    Phil S Baran; Fiscal Year: 2013
  2. DNA Assembly via Reiterative Recombination
    Virginia W Cornish; Fiscal Year: 2013
  3. Studies of Isoprenoid Biosynthesis
    CHARLES DALE POULTER; Fiscal Year: 2013
  4. New Methods and Strategies for the Synthesis of Anticancer Alkaloids
    RYAN ASHOK SHENVI; Fiscal Year: 2013
  5. Biosynthesis of Microbial Isoprenoids
    David E Cane; Fiscal Year: 2013
  6. Conservation and Sustainable Use of Biodiversity in PNG
    Louis R Barrows; Fiscal Year: 2013
  7. Conditional Expression of Mycobacterial Genes
    Sabine Ehrt; Fiscal Year: 2013
  8. Biosynthesis of marine polyketide antibiotics
    Bradley S Moore; Fiscal Year: 2013
  9. NMR studies of heterocyclization and epimerization in yersiniabactin synthesis
    DOMINIQUE PASCAL FRUEH; Fiscal Year: 2013
  10. Discovery of Natural Product based Drugs and Bioenergetic Materials from CR
    David H Sherman; Fiscal Year: 2013

Detail Information

Publications34

  1. pmc Synergistic substrate inhibition of ent-copalyl diphosphate synthase: a potential feed-forward inhibition mechanism limiting gibberellin metabolism
    Sladjana Prisic
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Plant Physiol 144:445-54. 2007
    ..Finally, the observed synergistic substrate/feed-forward inhibition of CPS also seems to provide a novel example of direct regulation of enzymatic activity in hormone biosynthesis...
  2. pmc Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase
    Mustafa Köksal
    Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA
    Nat Chem Biol 7:431-3. 2011
    ..Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions...
  3. pmc Domain loss has independently occurred multiple times in plant terpene synthase evolution
    Matthew L Hillwig
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Plant J 68:1051-60. 2011
    ....
  4. pmc Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis
    Ke Zhou
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
    J Biol Chem 287:6840-50. 2012
    ..Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities...
  5. pmc Effect of isotopically sensitive branching on product distribution for pentalenene synthase: support for a mechanism predicted by quantum chemistry
    Liansuo Zu
    Department of Chemistry, University of California, Davis, California 95616, USA
    J Am Chem Soc 134:11369-71. 2012
    ..These experimental results support a mechanism for pentalenene formation involving a 7-protoilludyl cation whose intermediacy was first predicted using quantum-chemical calculations...
  6. pmc Terpenoid synthase structures: a so far incomplete view of complex catalysis
    Yang Gao
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Nat Prod Rep 29:1153-75. 2012
    ..Accordingly, selected examples are discussed here to demonstrate our current understanding, its limits, and potential ways forward...
  7. pmc Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals
    Yisheng Wu
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Phytochemistry 84:40-6. 2012
    ..These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here...
  8. pmc Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals
    Ke Zhou
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Phytochemistry 84:47-55. 2012
    ....
  9. pmc Functional characterization and evolution of the isotuberculosinol operon in Mycobacterium tuberculosis and related Mycobacteria
    Francis M Mann
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University Ames, IA, USA
    Front Microbiol 3:368. 2012
    ....
  10. pmc A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase
    Jared Criswell
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
    Org Lett 14:5828-31. 2012
    ..Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity...
  11. pmc 1.55Å-resolution structure of ent-copalyl diphosphate synthase and exploration of general acid function by site-directed mutagenesis
    Mustafa Köksal
    Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104 6323, USA
    Biochim Biophys Acta 1840:184-90. 2014
    ..25Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αβγ domain architecture, but ambiguities regarding substrate analog binding remained...
  12. pmc Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae
    Jiachen Zi
    Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Org Biomol Chem 11:7650-2. 2013
    ....
  13. pmc To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism
    Jiachen Zi
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 email
    Annu Rev Plant Biol 65:259-86. 2014
    ..It additionally addresses the array of biological activity providing the selective pressures that drive the observed gene family expansion and diversification, along with biosynthetic gene clustering...
  14. pmc Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization
    Alana J Jackson
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Phytochemistry 103:13-21. 2014
    ..Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway. ..
  15. pmc Evident and latent plasticity across the rice diterpene synthase family with potential implications for the evolution of diterpenoid metabolism in the cereals
    Dana Morrone
    Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Biochem J 435:589-95. 2011
    ..e. normal CPP) which may arise during evolution, the implications of which are discussed...
  16. pmc Electrostatic effects on (di)terpene synthase product outcome
    Ke Zhou
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Chem Commun (Camb) 47:4074-80. 2011
    ..Thus, (di)terpene synthases seem to mediate specific reaction outcomes, at least in part, by providing electrostatic effects to counteract those exerted by the pyrophosphate co-product...
  17. pmc Following evolution's lead to a single residue switch for diterpene synthase product outcome
    Meimei Xu
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Proc Natl Acad Sci U S A 104:7397-401. 2007
    ....
  18. ncbi Probing the role of the DXDD motif in Class II diterpene cyclases
    Sladjana Prisic
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Molecular Biology Building, Ames, IA 50011, USA
    Chembiochem 8:869-74. 2007
  19. pmc A modular approach for facile biosynthesis of labdane-related diterpenes
    Anthony Cyr
    Iowa State University, Department of Biochemistry, Biophysics, and Molecular Biology, Ames, Iowa 50011, USA
    J Am Chem Soc 129:6684-5. 2007
  20. pmc 16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana
    Arnab Roy
    Department of Chemistry University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA
    J Am Chem Soc 129:12453-60. 2007
    ....
  21. pmc A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase
    P Ross Wilderman
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
    J Am Chem Soc 129:15736-7. 2007
    ..Furthermore, these defined single residue switches may be useful in engineering product outcome in diterpene synthases more generally...
  22. ncbi Increasing complexity of a diterpene synthase reaction with a single residue switch
    Dana Morrone
    Iowa State University, Department of Biochemistry, Biophysics, and Molecular Biology, Ames, Iowa 50011, USA
    J Am Chem Soc 130:5400-1. 2008
    ..Furthermore, the profound plasticity of diterpene synthases exemplified by this single residue switch for product outcome is consistent with the screening/diversity-oriented hypothesis of natural products metabolism...
  23. pmc Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants
    Ke Zhou
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Molecular Biology Building, Rm 4216, Ames, IA 50011, United States
    Phytochemistry 70:366-9. 2009
    ..In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS...
  24. pmc Characterization and inhibition of a class II diterpene cyclase from Mycobacterium tuberculosis: implications for tuberculosis
    Francis M Mann
    Department of Biochemistry, Iowa State University, Ames, Iowa 50011, USA
    J Biol Chem 284:23574-9. 2009
    ..Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans...
  25. pmc Edaxadiene: a new bioactive diterpene from Mycobacterium tuberculosis
    Francis M Mann
    Iowa State University, Department of Biochemistry, Biophysics, and Molecular Biology, Ames, Iowa 5001, USA
    J Am Chem Soc 131:17526-7. 2009
    ....
  26. pmc A single residue switch for Mg(2+)-dependent inhibition characterizes plant class II diterpene cyclases from primary and secondary metabolism
    Francis M Mann
    From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
    J Biol Chem 285:20558-63. 2010
    ....
  27. pmc Synthesis of (+/-)-nosyberkol (isotuberculosinol, revised structure of edaxadiene) and (+/-)-tuberculosinol
    Nathan Maugel
    Department of Chemistry MS 015, Brandeis University, Waltham, Massachusetts 02454 9110, USA
    Org Lett 12:2626-9. 2010
    ..The spectral data for nosyberkol are identical with those reported for edaxadiene, whose structure is revised accordingly...
  28. pmc Diterpene cyclases and the nature of the isoprene fold
    Rong Cao
    Center for Biophysics and Computational Biology, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Proteins 78:2417-32. 2010
    ..The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (betagamma) bacterial triterpene cyclases to (betagamma) bacterial and thence to (alphabetagamma) plant diterpene cyclases...
  29. pmc Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis
    Francis M Mann
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    FEBS Lett 585:549-54. 2011
    ..Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis...
  30. pmc Magnesium depletion triggers production of an immune modulating diterpenoid in Mycobacterium tuberculosis
    Francis M Mann
    Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA
    Mol Microbiol 79:1594-601. 2011
    ..These results demonstrate isoTb production by Mtb specifically under conditions that mimic phagosomal cation concentrations, and further support a role for isoTb in the Mtb infection process...
  31. pmc Novel product chemistry from mechanistic analysis of ent-copalyl diphosphate synthases from plant hormone biosynthesis
    Kevin Potter
    Dept Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 USA
    Angew Chem Int Ed Engl 53:7198-202. 2014
    ..Given the requisite presence of CPSs in all land plants for gibberellin phytohormone biosynthesis, such plasticity presumably underlies the observed extensive diversification of the resulting labdane-related diterpenoids. ..

Research Grants30

  1. Synthesis of Complex Terpenes
    Phil S Baran; Fiscal Year: 2013
    ....
  2. DNA Assembly via Reiterative Recombination
    Virginia W Cornish; Fiscal Year: 2013
    ....
  3. Studies of Isoprenoid Biosynthesis
    CHARLES DALE POULTER; Fiscal Year: 2013
    ....
  4. New Methods and Strategies for the Synthesis of Anticancer Alkaloids
    RYAN ASHOK SHENVI; Fiscal Year: 2013
    ..Auxiliary and catalyst-control over stereoselective thiaspirane formation will be investigated to rapidly synthesize al four stereoisomers and all sixteen possible family members of this fascinating class. ..
  5. Biosynthesis of Microbial Isoprenoids
    David E Cane; Fiscal Year: 2013
    ....
  6. Conservation and Sustainable Use of Biodiversity in PNG
    Louis R Barrows; Fiscal Year: 2013
    ..Legislative initiatives developed with government input promote scientific exchange, conservation and sustainable resource use. ..
  7. Conditional Expression of Mycobacterial Genes
    Sabine Ehrt; Fiscal Year: 2013
    ..Our work will provide a generally applicable blue print for the systematic evaluation of metabolic enzymes and pathways in vitro and in vivo and identify the glycolytic and gluconeogenic enzymes most attractive for drug development. ..
  8. Biosynthesis of marine polyketide antibiotics
    Bradley S Moore; Fiscal Year: 2013
    ..And fourth, we will functionally characterize the biosynthesis of the marinopyrrole antibiotics and their novel bipyrrole coupling enzymology. ..
  9. NMR studies of heterocyclization and epimerization in yersiniabactin synthesis
    DOMINIQUE PASCAL FRUEH; Fiscal Year: 2013
    ....
  10. Discovery of Natural Product based Drugs and Bioenergetic Materials from CR
    David H Sherman; Fiscal Year: 2013
    ..g., thioester hydrolases and decarboxylases) with application in liquid fuel production (biodiesel). ..
  11. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  12. DRUGS OF ABUSE - ROLE OF PROTEIN PHOSPHORYLATION
    Paul Greengard; Fiscal Year: 2013
    ..Results from the three Projects will complement each other. In addition, there will be a significant level of collaboration between the three Projects, as well as close interaction of the three Projects with the Scientific Core. ..
  13. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  14. TOTAL SYNTHESIS AND BIOSYNTHESIS OF BIOACTIVE SUBSTANCES
    Robert Michael Williams; Fiscal Year: 2013
    ..In addition, the chemical technologies being developed, will be applied to the design and synthesis of new anti-cancer drugs. ..
  15. Targeting Non-mammalian Isoprenoid Biosynthesis
    CAREN L MEYERS; Fiscal Year: 2013
    ..Anti-infective agents developed to target these enzymes have the potential to broadly impact the treatment of deadly infectious diseases. ..
  16. Fungal Polyketide Synthases and Environmental Toxin Biosynthesis
    CRAIG ARTHUR TOWNSEND; Fiscal Year: 2013
    ....