Stereochemistry of Δ<sup>4</sup>dehydrogenation catalyzed by an ivy (Hedera helix) Δ<sup>9</sup>desaturase homolog

Tremblay, Amy E.; Whittle, Edward; Buist, Peter H.; Shanklin, John

doi:10.1039/b617942h

Cited by 12 publications

(11 citation statements)

References 35 publications

(23 reference statements)

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“…This orientation of ACP could precisely orient the acyl chain for Δ4 desaturation of 16∶0 (Fig. 4B) with the previously reported pro-R pro-R enantioselectivity (15). Using the castor enzyme, with Asp280 exchanged for a lysine residue in the modeling, gave the same approximate orientation, which prompted us to probe the potential role of Asp280 for regioselectivity.…”

Section: Docking Of Acp To the Ivy Desaturase Identifies An Alternatimentioning

confidence: 98%

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Guy

Whittle

Moche

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase sidechain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.diiron enzyme | enzyme redesign | lipid metabolism | enzyme mechanism A cyl-acyl carrier protein (ACP) desaturases are a family of diiron center containing soluble enzymes that introduce double bonds into acyl-ACPs in an oxygen-dependent reaction. In plants, acyl-ACP desaturases play a key role in biosynthesis of monounsaturated fatty acids, acting as the primary determinant for the composition of unsaturated fatty acids in plant membranes and seed oils. A striking feature of the reaction catalyzed by these enzymes is that it is performed with extremely high stereo-and regioselectivity, despite the fact that the substrates are composed of essentially equivalent methylene chains that completely lack distinguishing landmarks close to the site of desaturation. As Nobel Prize winner Konrad Bloch observed in 1969, "The stereospecific removal of hydrogen in the formation of oleate, although predictable on principle grounds would seem to approach the limits of the discriminatory power of enzymes" (1).We have a long-standing interest in identifying the bases of this discriminatory power of desaturases. Acyl-ACP desaturases are Δ counters-i.e., they insert a double bond a certain number of carbons from the carboxyl end of the fatty acid (2). A detailed understanding of how this counting occurs could enable reengineering of ...

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Section: Docking Of Acp To the Ivy Desaturase Identifies An Alternatimentioning

confidence: 98%

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Guy

Whittle

Moche

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Some fundamental differences exist between the soluble and microsomal enzymes. For example, the cryptoregiochemistry of the hydrogen abstraction in the soluble Δ 9 -desaturase family, which favors abstraction of the pro-(R) hydrogen distal to the carbonyl group (e.g., H-10R in castor desaturases) independent of regiochemistry [114], is opposite to the membrane desaturases, which remove the pro-(R) hydrogen proximal to the carbonyl group. The model currently used for the active site is that of a compound Q-like oxidant based on the more developed enzymology of soluble methane monooxygenase [115].…”

Section: Cloning and Expression Of The Lipid-desaturating Components Ofmentioning

confidence: 99%

Biosynthesis and function of polyacetylenes and allied natural products

Minto

Blacklock

2008

Progress in Lipid Research

297

228

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Polyacetylenic natural products are a substantial class of often unstable compounds containing a unique carbon-carbon triple bond functionality, that are intriguing for their wide variety of biochemical and ecological functions, economic potential, and surprising mode of biosynthesis. Isotopic tracer experiments between 1960 and 1990 demonstrated that the majority of these compounds are derived from fatty acid and polyketide precursors. During the past decade, research into the metabolism of polyacetylenes has swiftly advanced, driven by the cloning of the first genes responsible for polyacetylene biosynthesis in plants, moss, fungi, and actinomycetes, and the initial characterization of the gene products.The current state of knowledge of the biochemistry and molecular genetics of polyacetylenic secondary metabolic pathways will be presented together with an up-to-date survey of new terrestrial and marine natural products, their known biological activities, and a discussion of their likely metabolic origins.

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“…Because the ivy desaturase introduces a ⌬ 4 double bond into a 16:0-substrate, five carbons distant from that of the castor desaturase with the equivalent substrate, and with the same Pro-R Pro-R stereoselectivity as the castor enzyme (22), we reason that differences in substrate binding between the two enzymes must account for differences in observed regioselectivity. We have initiated x-ray crystallographic analysis of the ivy desaturase to address this hypothesis.…”

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The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

Guy

Whittle

Kumaran

et al. 2007

Journal of Biological Chemistry

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The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the ⌬ 4 desaturation of 16:0-ACP and the ⌬ 9 desaturation of 18:0-ACP and further desaturates ⌬ 9 -16:1 or ⌬ 9 -18:1 to the corresponding ⌬ 4,9 dienes. The crystal structure of the enzyme has been solved to 1.95 Å resolution, and both the iron-iron distance of ϳ3.2 Å and the presence of a -oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His 227 coordination to Fe2 is lost, and the side chain of Glu 224 , which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site. Fatty acid acyl carrier protein (ACP)3 desaturases (EC 1.14.99.6) convert saturated fatty acyl-ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction (1-6). ⌬ 9 -desaturation of 18:0-ACP 4 occurs ubiquitously in the plastids of plants and thus plays an important role in determining the fluidity of cell membranes (7). Plants can express distinct acyl ACP desaturases, e.g. Arabidopsis thaliana has a family of seven acyl-ACP desaturases that are differentially expressed in different tissues (8). A structurally related homolog with unknown function from Mycobacterium has also been described (9).Seed oils of various plants accumulate monounsaturated fatty acids with unusual chain lengths and/or double bond positions. Several ⌬ 9 desaturases that recognize 14-, 16-, and 18-carbon chain lengths (4, 10 -12) have been described, and others with ⌬ 6 and ⌬ 9 regiospecificities have also been reported (13,14). The biosynthetic origin of unusual fatty acids is not always straightforward; for instance, Coriandrum sativum L. (coriander) and Hedera helix (English ivy) accumulate petroselinic acid (18:1⌬ 6 ) that originates via a ⌬ 4 desaturation of 16:0-ACP followed by 2-carbon elongation to 18:1⌬ 6 (15). The genes encoding these ⌬ 4 -16:0-ACP desaturase enzymes have been isolated (15,16), and their amino acid sequences are homologous to that of the castor ⌬ 9 -18:0-ACP desaturase for which the crystal structure has been determined (17,18). This implies that ac...

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Stereochemistry of Δ⁴dehydrogenation catalyzed by an ivy (Hedera helix) Δ⁹desaturase homolog

Cited by 12 publications

References 35 publications

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Biosynthesis and function of polyacetylenes and allied natural products

The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

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Stereochemistry of Δ4dehydrogenation catalyzed by an ivy (Hedera helix) Δ9desaturase homolog

Cited by 12 publications

References 35 publications

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Biosynthesis and function of polyacetylenes and allied natural products

The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

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Stereochemistry of Δ⁴dehydrogenation catalyzed by an ivy (Hedera helix) Δ⁹desaturase homolog