John Shanklin Senior Biochemist Biochemistry of Lipid Modification Enzymes Biology Department, 463 Brookhaven National Laboratory Upton, NY 11973-5000 tel: (631) 344-3414 fax: (631) 344-3407 shanklin@bnl.gov The Group: John Shanklin 344-3414 Carl Andre 344-5360 Ying Lou 344-5360 Girish Mishra 344-5360 Tam Nguyen 344-5360 Edward J. Whittle 344-5360

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 and John Ohlrogge      with Ian and Anona Gilliard.

Selected References:

Note: Selected PDB files below can be viewed with

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. (in press).(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: 10266-71 (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   Jmol viewer

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. andx 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

Related References - describing the above work

Browse, J.B., Towards rational engineering of plant oils: crystal structure of the 18:0-ACP desaturase. Trends in Plant Science 1(12), 403-404 (1996).

Raloff, J., A giant step toward creating better fats. Science News 151, 335 (1997).

Raloff, J., Genetically engineering a healthier margerine. Science News Online.

Updated Feb. 14, 2008	Security Notice   Webteam   Site Map   Biology Home   BNL Home