General Information

Top of Page

John Shanklin

Biochemistry of Lipid Modification Enzymes

As the population of the world increases there are increasing demands on natural resources. One solution to this problem is to use plants as 'Green Factories' to produce specific renewable resources and sources of bioenergy. Advances in genetic engineering have resulted in routine methods for the introduction of genes into crop plants. Previous efforts in crop improvement have focused on the transfer of existing genes into plants to tailor plant storage compounds. A key element of future efforts will be directed towards engineering enzymes with novel specificities and/or the ability to introduce a particular functionality. This will free metabolic engineers from the constraints imposed by the existing variation of natural enzymes and will present a major step towards the engineering of desired storage compounds.

Research Interests

Lipids are a major functional components of all living things. Despite their importance, the details of lipid synthesis and modification are incomplete. Lipids are first synthesized as saturated fatty acids and double bonds are incorporated post-synthetically by enzymes known as fatty acid desaturases; in a process that is initiated by cleavage of the stable methylene C-H bond. This reaction involves high energy chemistry, the details of which we are trying to elucidate. We are also interested in using this class of enzymes to further our understanding the basis for enzyme specificity and selectivity. Much of the tremendous diversity of fatty acids found in nature is due to the presence of variant desaturase-like enzymes. Desaturation of a fatty acid results in dramatic effects on the physical properties of both cell membranes and storage lipids, and the placement of double bonds, triple bonds, hydroxy groups, epoxy groups, and various conjugated double bond systems determines the uses to which these fatty acids can be put.

                     

Structural model of the soluble castor desaturase showing the location of several mutated residues at the bottom of the substrate binding cavity (left) and details of the oxidized diiron active site (right).

We are performing structure-function analysis on both the soluble and integral membrane classes of desaturases with the broad goal of understanding the basis for chain length specificity, regiospecificity and the control of functional outcome. Our goal is to develop sufficient understanding to be able to control all three parameters at will. If we are able to do that we will be able to engineer enzymes for the synthesis of fatty acids with any desired structure/characteristic. We are also interested in the ways enzyme functionality evolves, and have already been able to engineer desaturase to have desired properties in a redesigned enzyme that outperforms naturally occurring enzymes of the same specificity when engineered into transgenic plants.


  

   View down the central four
   helix bundle of the desaturase
   showing the active site diiron
   center (orange spheres)and
   the amino acid side chains
   that play important roles
   in coordinating the diiron
   center and catalysis.


   Our model system for studying the soluble class of desaturases is the plant D9 stearoyl-ACP desaturase. Our approach involves the use of molecular, biochemical crystallography tools in addition to combinatorial genetic methods such as directed evolution. In addition we employ chiral labeled fatty acids as chemical probes of the reaction mechanism. We are currently investigating a series of positional- and chain length- specific isoforms of this enzyme from other plants to determine the structural basis of enzyme specificity. We have identified a variant soluble desaturase that hydroxylates fatty acids to produce allylic alcohols and are in the process of determining its reaction mechanism.

   A second, related project, focuses on the characterization of active site components of the major class of fatty acid desaturases that are integral membrane proteins. In recent studies we have defined the determinants of functional outcome in terms of desaturation or hydroxylation for this class of enzymes. Current work focuses on determining the crystal structure of AlkB, a member of this class of enzymes.




  Terry Galliard medal winners,
  John Shanklin, Norio Murata,
  John Harwood, John Ohlrogge,
  Ian and Anona Gilliard.



Awards:

  • Terry Galliard Medal for Plant Lipid Biochemistry; presenter Galliard Lecture at the 12th International Symposium on Plant Lipids, Toronto, Canada, July, 1996 DuPont Science and Engineering Educational Aid Grant, 1996 U.S. Department of Energy Office of Energy Research Young Scientist Award 1997
  • Presidential Early Career Award for Scientists and Engineers, 1997-2002
  • Nathan Edward Tolbert Endowed Lectureship Award, Department of Biochemistry and Molecular Biology, Michigan State University, May 3, 2001
  • U.S. Department of Energy Office of Science Pollution Prevention Award, 2004 Fellow of the American Association for the Advancement of Science (AAAS), 2008

Membership:

  • International Society for Plant Molecular Biology
  • American Society of Plant Biology
  • International Committee on Proteolysis
  • American Chemical Society
  • National Plant Lipid Cooperative (Program Committee Chair 2003-2006)

Advisory Board:

  • Genetic Engineering, Principles and Methods (book published annually by Plenum Press, 1996-Present)

Editorial Board:

  • Advances in Plant Biochemistry and Molecular Biology (2002-Present)

Recent News

Selected Publications

  • Wang W., Haberer G., Gundlach H., Gläβer C., Nussbaumer T., Luo M.C., Lomsadze A., Borodovsky M., Kerstetter R.A., Shanklin J., Bryant D.W., Mockler T.C., Appenroth K.J., Grimwood J., Jenkins J., Chow J., Choi C., Adam C., Cao X.-H., Fuchs J., Schubert I., Rokhsar D., Schmutz J., Michael T.P., Mayer K.F.X., and Messing J. The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nature Communications (Epub February 19, 2014).
  • Yu, X.-H., Prakash, R. R., Sweet, M., and Shanklin, J. Coexpressing Escherichia coli cyclopropane synthase with Sterculia foetida lysophosphatidic acid acyltransferase enhances cyclopropane fatty acid accumulation. Plant Physiol., 164(1):455-465 (2014).
  • Yan Y., Candreva J., Shi H., Ernst E., Martienssen R., Schwender J., and Shanklin J. Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba. BMC Plant Biology, 13:201 (2013).
  • Andre C., Kim S.-W., Yu X.-H., and Shanklin J. Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc. Natl. Acad. Sci. USA 110(8):3191-3196 (2013).
  • Cooper H.L.R., Mishra G., Huang X., Pender-Cudlip M., Austin R.N., Shanklin J., Groves J.T. Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB. J. Am. Chem. Soc., 134(50):20365-20375 (2012).
  • Andre C., Haslam R., and Shanklin J.
    Feedback regulation of plastidic Acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus.
    Proc. Natl. Acad. Sci. USA, 109(25):10107-10112 (2012).  PubMed
  • Fan J., Yan C., Andre C., Shanklin J., Schwender J., and Xu C.
    Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii.
    Plant Cell Physiol., 53(8): 1380-1390 (2012).  PubMed
  • Rawat R., Yu X.H., Sweet M., and Shanklin J.
    Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of the Momordica charantia conjugase.
    J. Biol. Chem., 287(20):16230-16237 (2012).  PubMed
  • Chen W., Yu X.-H., Zhang K., Shi J., Schreiber L., Shanklin J., and Zhang D.
    Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis.
    Plant Physiol., 157(2):842-853 (2011).   PubMed
  • Guy J.E., Whittle E., Moche M., Lengqvist J., Lindqvist Y., and Shanklin J.
    Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.
    Proc. Natl. Acad. Sci. USA, 108(40):16594-16599 (2011).  PubMed
  • Schluter P.M., Xu S., Gagliardini V. Whittle, E., Shanklin J., Grossniklaus U., and Schiestl F.P.
    Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids.
    Proc. Natl. Acad. Sci. USA, 108(14):5696-5701 (2011).  PubMed
  • Shi J., Tan H., Yu X.-H., Liu Y., Liang W., Ranathunge K., Franke R.B., Schreiber L., Wang Y., Kai G., Shanklin J., Ma H., and Zhang D.
    Defective Pollen Wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase.
    Plant Cell, 23(6):2225-2246 (2011).  PubMed
  • Yu X.-H., Rawat R., and Shanklin J.
    Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis.
    BMC Plant Biology, 11:97 (2011).   PubMed
  • Lou Y. and Shanklin J.
    Evidence that the yeast desaturase Ole1p exists as a dimer in vivo.
    J. Biol. Chem., 285(25):19384-19390 (2010).  PubMed
  • Nguyen H.T., Mishra G., Whittle E., Bevan S.A., Merlo A.O., Walsh T.A., and Shanklin J.
    Metabolic engineering of seeds can achieve levels of ω-7 fatty acids comparable to the highest levels found in natural plant sources.
    Plant Physiol., 154(4):1897-1904 (2010).  PubMed
  • Tremblay A.E., Tan N., Whittle E., Hodgson D.J., Dawson B., Buist P.H., and Shanklin J.
    Stereochemistry of 10-sulfoxidation catalyzed by a soluble Δ9 desaturase.
    Organic & Biomolecular Chemistry, 8(6):1322-1328 (2010).  PubMed
  • Nguyen H.T. and Shanklin J.
    Altering Arabidopsis oilseed composition by a combined antisense-hairpin RNAi gene suppressing approach.
    J. Amer. Oil Chemists Soc., 86(1):41-49 (2009).
  • Shanklin J., Guy J.E., Mishra G., and Lindqvist Y.
    Desaturases: Emerging models for understanding functional diversification of diiron-containing enzymes.
    Journal of Biological Chemistry 284(28): 18559-18563 (July, 2009).
    Cover: View down the four-helix bundle to the dirron active site of the castor desaturase mutant T199D, which displays decreased desaturation activity and increased oxidase functionality consistent with that of its hypothesized ancestor. The non-coordinating residue on the lower left is Asp199. For details see the article by Shanklin et al., pages 18559-18563.  PubMed
  • Shanklin J.
    Enzyme engineering.
    Advances in Plant Biochemistry and Molecular Biology, Vol. 1: Bioengineering and Molecular Biology of Plant Pathways, H. J. Bohnert, H. Nguyen, and N. Lewis, Editors, Chapter 2, Elsevier Science Ltd., Oxford, U.K. (2008).
  • Whittle E.J., Tremblay A.E., Buist P.H. and Shanklin J.
    Revealing the catalytic potential of an acyl-ACP desaturase: Tandem selective oxidation of saturated fatty acids.
    Proceedings of the National Academy of Sciences, 105(38):14738-14743 (2008).  PubMed
  • Zhang P., Burton J.W., Upchurch R.G., Whittle E., Shanklin J. and Dewey R.E.
    Mutations in a Δ9-stearoyl-ACP-desaturase gene are associated with enhanced stearic acid levels in soybean seeds.
    Crop Science, 48:2305-2313 (November-December, 2008).
  • Guy J.E., Whittle E., Kumaran D., Lindqvist Y. and Shanklin J.
    The crystal structure of the ivy Δ4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity.
    J. Biol. Chem., 282(27):19863-19871 (2007).   PubMed
  • Kachroo A., Shanklin J., Whittle E., Lapchyk L., Hildebrand D. and Kachroo P.
    The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis.
    Plant Molec. Biol., 63(2):257-271 (2007).   PubMed
  • Mayer K.M. and Shanklin J.
    Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using bioinformatics-guided approach.
    BMC Plant Biol., 7:1 (2007).   PubMed
  • Pidkowich M.S., Nguyen H.T., Heilmann I., Ischebeck T. and Shanklin J.
    Modulating seed β-ketoacyl-acyl carrier protein synthase II level converts the composition of a temperate seed oil to that of a palm-like tropical oil.
    Proc. Natl. Acad. Sci. USA, 104(11):4742-4747 (2007).   PubMed
  • Shanklin J. and Whittle E.
    Acyl-ACP desaturase architecture favors high-energy desaturation reaction over lower-energy oxidase chemistry.
    Current Advances in the Biochemistry and Cell Biology of Plant Lipids, Proceedings of the 17th International Symposium on Plant Lipids, July 16-21, 2006, Michigan State University, East Lansing, MI, C. Benning and J. Ohlrogge, Editors, pp. 207-209, Aardvark Global Publishing Company, LLC, Salt Lake City, UT. (2007).  
  • Tremblay A.E., Whittle E., Buist P.H. and Shanklin J.
    Stereochemistry of Δ4 dehydrogenation catalyzed by a Δ9 desaturase homolog.
    Organic Biomolec. Chem., 5(8):1270-1275 (2007).   PubMed
  • Guy J.E., Abreu I.A., Moche M., Lindqvist Y., Whittle E. and Shanklin J.
    A single mutation in the castor Δ9-18:0-desaturase changes reaction partitioning from Desaturation to oxidase chemistry.
    Proc. Natl. Acad. Sci. USA, 103(46):17220-17224 (2006).   PubMed
  • Tremblay A.E., Buist P.H., Hodgson D., Dawson B., Whittle E. and Shanklin J.
    In vitro enzymatic oxidation of a fluorine-tagged sulfide substrate analogue: A 19F NMR investigation.
    Magnetic Resonance in Chemistry, 44(6):629-632 (2006).  PubMed
  • Davydov R., Behrouzian B., Smoukov S., Stubbe J., Hoffman B.M., and Shanklin J.
    Effect of substrate on the diiron(III) site in stearoyl acyl carrier protein delta(9)-desaturase as disclosed by cryoreduction electron paramagnetic resonance/electron nuclear double resonance spectroscopy.
    Biochemistry, 44:1309-1315 (2005).   PubMed
  • Mayer K.M., McCorkle S.R. and Shanklin J.
    Linking enzyme sequence to function using conserved property difference locator to identify and annotate positions likely to control specific functionality.
    BMC Bioinformatics, 6:284 (2005). PubMed
  • Mayer KM, and Shanklin J.
    A structural model of the plant acyl-acyl carrier protein thioesterase FatB comprises two helix/4-stranded sheet domains, the N-terminal domain containing residues that affect specificity and the C-terminal domain containing catalytic residues.
    J. Biol. Chem., 280:3621-3627 (2005).   PubMed
    PDB file: 1XXY
  • Whittle E., Cahoon E.B., Subrahmanyam S. and Shanklin J.
    A multifunctional acyl-acyl carrier protein desaturase from Hedera helix L. (English ivy) can synthesize 16- and 18-carbon monoene and diene products.
    J. Biol Chem. June 6,(2005). Epub ahead of print   PubMed   Full Text
  • Heilmann I., Mekhedov S., King B., Browse J. and Shanklin J.
    Identification of the Arabidopsis palmitoyl-monogalactosyldiacylglycerol delta7-desaturase gene FAD5, and effects of plastidial retargeting of Arabidopsis desaturases on the fad5 mutant phenotype.
    Plant Physiol., 136:4237-4245 (2004).   PubMed
  • Heilmann I., Pidkowich M.S., Girke T. and Shanklin J.
    Switching desaturase enzyme specificity by alternate subcellular targeting.
    Proc Natl Acad Sci USA, 101:10266-10271 (2004).   PubMed   Full Text
    For a summary, read the BNL Press Release of 6 July, 2004.
  • Reipa V., Shanklin J. and Vilker V.
    Substrate binding and the presence of ferredoxin affect the redox properties of the soluble plant Delta9-18:0-acyl carrier protein desaturase.
    Chem. Commun. (Camb)., 21:2406-2407 (2004).   PubMed
  • Moche M., Shanklin J., Ghoshal A. and Lindqvist Y.
    Azide and acetate complexes plus two iron-depleted crystal structures of the di-iron enzyme delta 9 stearoyl-ACP desaturase-implications for oxygen activation and catalytic intermediates.
    J. Biol. Chem., 278:25072-25080 (2003).   PubMed   Full Text
    PDB files: 1OQ4, 1OQ7, 1OQ9, 1OQB  
  • Shanklin J. and Whittle E.
    Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.
    FEBS Letters, 545:188-192 (2003).   PubMed
  • Behrouzian B., Hodgson D., Savile C.K., Dawson B., Buist P.H. and Shanklin J.
    Use of 19F NMR spectroscopy to probe enzymatic oxidation of fluorine-tagged sulfides.
    Magn. Reson. Chem., 40:524-528 (2002).
  • Behrouzian B., Savile C.K., Dawson B., Buist P.H. and Shanklin J.
    Exploring the hydroxylation-dehydrogenation connection: Novel catalytic activity of castor stearoyl-ACP delta 9 desaturase.
    J. Amer. Chem. Soc., 124:3277-3283 (2002).   PubMed
  • Broadwater J.A., Whittle E. and Shanklin J.
    Desaturation and hydroxylation: Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity.
    J. Biol. Chem., 277:15613-15620 (2002).   PubMed   Full Text
  • Behrouzian B., Buist P.H. and Shanklin J.
    Application of KIE and thia approaches in the mechanistic study of a plant stearoyl-ACP delta-9 desaturase.
    Chemical Communications, 401-402 (2001).
  • Behrouzian B., Dawson B., Buist P.H. and Shanklin J.
    Oxidation of chiral 9-fluorinated substrates by castor stearoyl-ACP delta-9 desaturase yields novel products.
    Chemical Communications, 765-766 (2001).
  • Kachroo P., Shanklin J., Shah J., Whittle E.J. and Klessig D.F.
    A fatty acid desaturase modulates the activation of defense signaling pathways in plants.
    Proc. Natl. Acad. Sci. USA, 98:9448-9453 (2001).   PubMed   Full Text
  • Whittle E. and Shanklin J.
    Engineering delta-9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta-9-18:0-ACP desaturase.
    J. Biol. Chem., 276:21500-21505 (2001).   PubMed   Full Text
  • Cahoon E.B. and Shanklin J.
    Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils.
    Proc. Natl. Acad. Sci. USA, 97:12350-12355 (2000).   PubMed   Full Text
  • Shanklin J.
    Exploring the possibilities presented by protein engineering.
    Curr. Opin. Plant Biol., 3:243-248 (2000).   PubMed
  • Shanklin J.
    Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form.
    Protein Expr. Purif., 18:355-360 (2000).   PubMed
  • Broun P., Shanklin J., Whittle E. and Somerville C.
    Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids.
    Science, 282:1315-1317 (1998).   PubMed   Full Text
  • Cahoon E.B., Shah S., Shanklin J. and Browse J.
    A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed.
    Plant Physiol., 117:593-598 (1998).   PubMed   Full Text
  • Shanklin J. and Cahoon E.B.
    Desaturation and related modifications of fatty acids
    Annu Rev Plant Physiol Plant Mol Biol, 49:611-641 (1998).   PubMed
  • Shanklin J., Cahoon E.B. and Whittle E.
    Strategies for altering chain length specificity of acyl-ACP desaturases.
    in: Advances in Plant Lipid Research, Sanchez J., Cerda-Olmedo E., and Martinez-Force E., Editors, pp. 115-117, Secretariado de Publicaciones de la Universidad de Sevilla, Spain (1998).
  • Cahoon E.B., Coughlan S. and Shanklin J.
    Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed.
    Plant Mol. Biol., 33:1105-1110 (1997).   PubMed
  • Cahoon E.B., Lindqvist Y., Schneider G. and Shanklin J.
    Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position.
    Proc. Natl. Acad. Sci. USA, 94:4872-4877 (1997).   PubMed   Full Text
  • Cahoon E.B. and Shanklin J.
    Approaches to the design of acyl-ACP desaturases with altered fatty acid chain-length and double bond positional specificities.
    in: Physiology, Biochemistry and Molecular Biology of Plant Lipids, J.P. Williams, M.U. Khan and N.W. Lem, edts., pp 374-376, Kluwer Academic Publishers, The Netherlands, (1997).
  • Shanklin J., Achim C., Schmidt H., Fox B.G. and Münck E.
    Mössbauer studies of alkane omega-hydroxylase: Evidence for a diiron cluster in an integral membrane enzyme.
    Proc. Natl. Acad. Sci. USA, 94:2981-2986 (1997).   PubMed   Full Text
  • Shanklin J., Cahoon E.B., Whittle E.J., Lindqvist Y., Schneider G. and Schmidt H.
    Structure-function studies on desaturases and related hydrocarbon hydroxylases.
    in: Physiology, Biochemistry and Molecular Biology of Plant Lipids, J.P. Williams, M.U. Khan and N.W. Lem, edts., pp 6-10, Kluwer Academic Publishers, The Netherlands, (1997).
  • Cahoon E.B., Mills L.A. and ShanklinJ.
    Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl-acyl carrier protein desaturase and ferredoxin.
    J. Bacteriol., 178:936-939 (1996).   PubMed   Full Text
  • Lindqvist Y., Huang W., Schneider G. and Shanklin J.
    Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.
    EMBO J., 15:4081-4092 (1996).   PubMed
  • Schultz D.J., Cahoon E.B., Shanklin J., Craig R., Cox-Foster D.L., Mumma R.O. and Medford J.I.
    Expression of a delta9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega-5 anacardic acids found in pest-resistant geranium (Pelargonium xhotorum).
    Proc. Natl. Acad. Sci. USA, 93:8771-8775 (1996).   PubMed   Full Text
  • Flanagan J.M., Wall J.S., Capel M.S., Schneider D.K. and Shanklin J.
    Scanning transmission electron microscopy and small-angle scattering provide evidence that native Escherichia coli ClpP is a tetradecamer with an axial pore.
    Biochemistry, 34:10910-10917 (1995).   PubMed
  • Shanklin J., DeWitt N.D. and FlanaganJ.M.
    The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: An archetypal two-component ATP-dependent protease.
    Plant Cell, 7:1713-1722 (1995).   PubMed
  • Cahoon E.B., Cranmer A.M., Shanklin J. and Ohlrogge J.B.
    delta6 hexadecanoic acid is synthesized by the activity of a soluble delta6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.
    J. Biol. Chem., 269:27519-27526 (1994).   PubMed
  • Fox B.G., Shanklin J., Ai J., Loehr T.M. and Sanders-Loehr J.
    Resonance Raman evidence for an Fe-O-Fe center in stearoyl-ACP desaturase. Primary sequence identity with other diiron-oxo proteins.
    Biochemistry, 33:12776-12786 (1994).   PubMed
  • Shanklin J., Whittle E. and Fox B.G.
    Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase.
    Biochemistry, 33:12787-12794 (1994).   PubMed