Exploring the Hydroxylation−Dehydrogenation Connection:  Novel Catalytic Activity of Castor Stearoyl-ACP Δ<sup>9</sup> Desaturase

Behrouzian, Behnaz; Savile, Christopher K.; Dawson, Brian; Buist, Peter H.; Shanklin, John

doi:10.1021/ja012252l

Cited by 58 publications

(89 citation statements)

References 37 publications

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“…Although a crystal structure of the desaturase-substrate complex has yet to be published, modeling the substrate into the existing crystal structure suggests that when the methyl group of the stearate is in contact with the bottom of the cavity, C9 and C10 are positioned with their pro-R hydrogens facing the diiron active site (6). This structural model is consistent with the Pro-R Pro-R stereochemistry reported for the desaturase reaction (8) and the location of azide, a peroxo mimic, complexed with the desaturase diiron site (7).…”

supporting

confidence: 74%

A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

Whittle

Cahoon

Subrahmanyam

et al. 2005

Journal of Biological Chemistry

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A desaturase with 83% sequence identity to the coriander ⌬ -ACP to the corresponding ⌬ 4,9 dienes. These data suggest at least two distinct substrate binding modes, one placing C4 at the diiron active site and the other placing C9 at the active site. In the latter case, 18:0 would likely bind in an extended conformation as described for the castor desaturase with 9-carbons accommodated in the cavity beyond the dirron site. However, ⌬ 4 desaturation would require the accommodation of 12 carbons for C16 substrates or 14 carbons for C18 substrates. The amino acids lining the substrate binding cavity of ivy and castor desaturases are conserved except for T117R and P179I (castor/ivy). Paradoxically, both substitutions, when introduced into the castor desaturase, favored the binding of shorter acyl chains. Thus, it seems likely that ⌬ 4 desaturation would require a non-extended, perhaps U-shaped, substrate conformation. A cis double bond may facilitate the initiation of such a non-extended conformation in the monounsaturated substrates. The multifunctional properties of the ivy desaturase make it well suited for further dissection of the determinants of regiospecificity. Acyl-acyl carrier protein (ACP)1 desaturases (EC 1.14.99.6) are responsible for the conversion of saturated fatty acyl-ACPs into monounsaturated-ACPs (1-3). This reaction is the penultimate step in the ACP track of fatty acid biosynthesis in the plastid and represents the major source of monounsaturated fatty acids in plant tissues (4). The resulting monounsaturated fatty acids are required to maintain membrane fluidity and in storage tissues are directed into storage triacylglycerols either directly or after additional desaturation steps. Plant acyl-ACP desaturases belong to a distinct sequence class composed of soluble globular proteins, whereas most fatty acid desaturases, found in fungi and animal and plants, are integral membrane proteins (5). All desaturases identified to date that recognize monoene, diene, and polyene substrates are members of the integral membrane class of desaturases. The archetypal ⌬ 9 -18: 0-ACP 2 desaturase from Ricinus communis (castor) has been the focus of extensive structure-function studies because it is readily expressed in active form in Escherichia coli, and it is the only member of the acyl-ACP desaturase family for which a three-dimensional crystal structure has been determined (6, 7). Each monomer of the castor enzyme homodimer contains a boomerang-shaped cavity capable of binding a stearoyl moiety in a gauche conformation adjacent to the active site diiron cluster. Although a crystal structure of the desaturase-substrate complex has yet to be published, modeling the substrate into the existing crystal structure suggests that when the methyl group of the stearate is in contact with the bottom of the cavity, C9 and C10 are positioned with their pro-R hydrogens facing the diiron active site (6). This structural model is consistent with the Pro-R Pro-R stereochemistry reported for the desaturase reaction (8) and th...

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confidence: 74%

A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

Whittle

Cahoon

Subrahmanyam

et al. 2005

Journal of Biological Chemistry

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“…The crystal structure showed the desaturase to be a homodimeric protein with a conserved four-helix bundle, containing the diiron active site, at the core of each monomer. The structure also revealed a substrate-binding cavity capable of binding the acyl chain in an extended configuration, within which a "boomerang-shaped" bend imparts an eclipsed substrate conformation, consistent with the observed pro-R pro-R dehydrogenation (6) which leads to the formation of the cis double bond. Although this structure explained the mechanism of stereoselectivity, the determinants of regioselectivity remained elusive.…”

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confidence: 63%

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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“…Second, enzymes would have to be capable of accepting two or more alternate substrates for catalysis. A survey of plant lipidmodifying enzymes alone yields many examples of bifunctional enzymes, including desaturases, hydroxylases, and conjugases (36,(40)(41)(42)(43)(44), suggesting that plants contain many bifunctional or multifunctional enzymes. Third, compartments would have to contain specific complements of metabolites, a condition that has been experimentally observed for many decades.…”

Section: Resultsmentioning

confidence: 99%

Switching desaturase enzyme specificity by alternate subcellular targeting

Heilmann

Pidkowich²,

Girke³

et al. 2004

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

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The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise. However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context. When the plastidial Arabidopsis 16:0⌬ 7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, ⌬ 7 to ⌬ 9 . Conversely, retargeting of two related cytoplasmic 16:0⌬ 9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity Ϸ25-fold, ⌬ 9 to ⌬ 7 . All three desaturases exhibited ⌬ 9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred ⌬ 7 desaturation, with 16:1⌬ 7 accumulating specifically on the plastidial lipid MGDG. Positional analysis is consistent with ADS desaturation of 16:0 on MGDG. The lipid headgroup acts as a molecular switch for desaturase regiospecificity. FAD5 ⌬ 7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic ⌬ 9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1⌬ 7 and its abundant metabolite, 16:3⌬ 7,10,13 . Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread. Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments.M etabolic diversity in living systems arises primarily from biotransformations catalyzed by enzymes; indeed life itself depends on the specificity of enzymes. The unique functionality, substrate specificity, and regiospecificity of an enzyme typically evolves by the gradual accumulation of changes in the catalytic portion of the enzyme until new properties arise. However, there is an emerging body of evidence suggesting that an enzyme's functional characteristics can also be affected by its metabolic context and that temporal and spatial dynamics of enzyme interactions can be important determinants of enzyme functionality (1). Examples include the ''rewiring'' of mitogen-activated protein kinase pathways on alternative scaffolds (2); the localization-dependent interactions of transcription factors with RNA polymerase in the nucleus (reviewed in ref. 3); the interchangeable tissue-specific roles of the transcription factors WER and GL1 in plant epidermal hair development (4); the modification of laccase͞peroxidase activity in the extracellular matrix by specific dirigent proteins (reviewed in ref. 5); the altered gati...

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Exploring the Hydroxylation−Dehydrogenation Connection: Novel Catalytic Activity of Castor Stearoyl-ACP Δ⁹ Desaturase

Cited by 58 publications

References 37 publications

A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

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

Switching desaturase enzyme specificity by alternate subcellular targeting

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Exploring the Hydroxylation−Dehydrogenation Connection: Novel Catalytic Activity of Castor Stearoyl-ACP Δ9 Desaturase

Cited by 58 publications

References 37 publications

A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

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

Switching desaturase enzyme specificity by alternate subcellular targeting

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Exploring the Hydroxylation−Dehydrogenation Connection: Novel Catalytic Activity of Castor Stearoyl-ACP Δ⁹ Desaturase