Identification of Non-Heme Diiron Proteins That Catalyze Triple Bond and Epoxy Group Formation

Lee, Michael A.; Lenman, Marit; Banaś, Antoni; Bafor, Maureen; Singh, Surinder P.; Schweizer, Michael; Nilsson, Ralf; Liljenberg, Conny; Dahlqvist, Anders; Gummeson, Per-Olov; Sjödahl, Staffan; Green, Allan; Stymne, Sten

doi:10.1126/science.280.5365.915

Cited by 244 publications

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“…By analogy to soluble fatty acid desaturases, the presence of conserved His boxes of the formulas HX [3][4] H, HX 2-3 HH, and (H/Q)X 2-3 HH has led to the suggestion that these include the ligands for a diiron cluster at the active site of the enzyme (6) (Figure 2). Amino acid residues that are proposed to contribute to activity tend to cluster around these His boxes (7-10) while hydropathy plots suggest four conserved transmembrane domains (11) (Figure 2).…”

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“…These reactions include hydroxylation, epoxidation, conjugated double bond formation, and triple bond formation (3). The acetylenase Crep1, from Crepis alpina, is the first of a number of FAD2-like acetylenases identified in the plant families Asteraceae, Apiaceae, and Araliaceae (4). Crep1 catalyzes the formation of a triple bond at the 12,13 position of linoleate (18:2-9c,12c) to give crepenynate (18:1-9c,12a) ( Figure 1).…”

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Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

et al. 2009

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Structural control of chemoselectivity, stereoselectivity, and substrate specificity in membrane-bound fatty acid acetylenases and desaturases Gagné, Steve J.; Reed, Darwin W.; Gray, Gordon R.; Covello, Patrick S. Saskatoon SK S7N 5A8, Canada Received September 14, 2009; Revised Manuscript Received October 26, 2009 ABSTRACT: The FAD2-like desaturases comprise a group of membrane-bound oxygenases involved in the modification of fatty acyl groups in plants and fungi. This group includes typical oleate desaturases which introduce a Δ12 cis double bond and more unusual enzymes such as Crep1, an acetylenase from the plant Crepis alpina, which introduces a triple bond in linoleate at the Δ12 position. In this study, the structure--function relationship between FAD2-like acetylenases and desaturases was examined through site-directed mutagenesis and heterologous expression. Eleven amino acid positions were identified that show complete evolutionary conservation within acetylenases or desaturases but have different amino acids in the other class of enzyme. Point mutants in Crep1 were constructed and expressed in yeast to test the role in fatty acid modification of the amino acids at the 11 positions. Results indicate the importance of five amino acid positions within Crep1 with regard to desaturase and acetylenase chemoselectivity, stereoselectivity, and substrate recognition. For example, relative to wild-type Crep1, the Y150F, F259L, and H266Q mutations all favored desaturation over acetylenation. The data indicate that small changes in primary sequence, particularly in the vicinity of the active site, can have profound changes on chemoselectivity and other aspects of the function of membrane-bound desaturase-like enzymes.The FAD2 1 -like desaturases comprise a group of membranebound oxygenases involved in the modification of fatty acyl groups in plants and fungi. The archetypal plant FAD2 catalyzes the introduction of a cis double bond at the Δ12 position of oleoylphosphatidylcholine in the endoplasmic reticulum (1) (Figure 1). This requires molecular oxygen and reducing equivalents from cytochrome b 5 (2).Interestingly, the FAD2-like enzyme group includes a number of variants which catalyze reactions other than the usual Δ12 cis desaturation. These reactions include hydroxylation, epoxidation, conjugated double bond formation, and triple bond formation (3). The acetylenase Crep1, from Crepis alpina, is the first of a number of FAD2-like acetylenases identified in the plant families Asteraceae, Apiaceae, and Araliaceae (4). Crep1 catalyzes the formation of a triple bond at the 12,13 position of linoleate (18:2-9c,12c) to give crepenynate (18:1-9c,12a) (Figure 1). Crep1 has also been reported to catalyze both cis and trans desaturation of oleate (5) (Figure 1).In general, the membrane-bound desaturases are particularly difficult to work with in vitro.Muchofwhatisknownaboutthe structure and function of these enzymes has been derived from their primary structure and in vivo activities in yeast. By analogy to solub...

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Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

et al. 2009

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“…This is the first reported synthesis of crepenynic acid in animals; the genes conducting the identical transformation in the plant C. alpina and the chanterelle mushroom C. formosus have been identified 7,8 , but have essentially no sequence similarity to the soldier beetle ∆12 desaturase/acetylenase gene or encoded protein.…”

Section: Figure 5 | Phylogenetic Analysis Of Insect Desaturasesmentioning

confidence: 99%

“…1) as the precursor fatty acid. Divergent isoforms of the microsomal acyl lipid-linked fatty acid desaturases introduce an acetylenic bond into the ∆12 position of linoleic acid to form crepenynic acid in the daisy Crepis alpina 7 and in the chanterelle mushroom Cantharellus formosus 8 . However, to date, advancement of this field has been restricted to the first acetylenic bond formation in the ∆12 position and subsequent ∆14 unsaturation of the polyacetylenic biosynthesis pathway 8 .…”

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“…However, to date, advancement of this field has been restricted to the first acetylenic bond formation in the ∆12 position and subsequent ∆14 unsaturation of the polyacetylenic biosynthesis pathway 8 . Fatty acid desaturases belong to a large family of non-heme diiron oxido-reduction enzymes that, in effect, remove two hydrogen atoms from adjacent carbons in a fatty acid chain to form a double bond, or in the case of acetylenases a further pair of hydrogens are removed from an existing double bond to produce a triple bond 7,9 . Integral-membrane desaturases are differentiated by their substrate preferences, either of the phospholipid-linked substrate preference found in plants and fungi, whereas acyl Coenzyme A (acyl-CoA)-linked preferences are mainly found in higher animals, including insects 10,11 .…”

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The convergent evolution of defensive polyacetylenic fatty acid biosynthesis genes in soldier beetles

et al. 2012

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The defensive and bioactive polyacetylenic fatty acid, 8Z-dihydromatricaria acid, is sequestered within a wide range of organisms, including plants, fungi and soldier beetles. The 8Z-dihydromatricaria acid is concentrated in the defence and accessory glands of soldier beetles to repel avian predators and protect eggs. In eukaryotes, acetylenic modifications of fatty acids are catalysed by acetylenases, which are desaturase-like enzymes that act on existing double bonds. Here we obtained acyl Coenzyme A-linked desaturases from soldier beetle RnA and functionally expressed them in yeast. We show that three genes were sufficient for the conversion of a common monounsaturated fatty acid, oleic acid, to the 18 carbon precursor of 8Z-dihydromatricaria acid, that is, 9Z,16Z-octadecadiene-12,14-diynoic acid. These are the first eukaryotic genes reported to produce conjugated polyacetylenic fatty acids. Phylogenetic analysis shows that the genes responsible for 8Z-dihydromatricaria acid synthesis in soldier beetles evolved de novo and independently of the acetylenases of plants and fungi.

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Lipid Metabolism

Schmid

2004

Handbook of Plant Biotechnology

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Lipids are an immensely diverse group of compounds, technically including all biomolecules that dissolve more readily in non‐polar solvents than in water. This review will emphasisze two prominent groups of lipids: the acyl lipids, whose hydrophobic units are derived from fatty acids, and the isoprenoids, made up of five‐carbon isopentene units. Acyl lipids, in the form of the triacylglycerols stored as energy reserves, underpin the vegetable oil industry. In addition, acyl lipids that dominate the bilayers of plant membranes, are indispensable for the protective layers that prevent plant desiccation and pathogen attack, and include several signalling molecules. Isoprenoids are even more diverse than acyl lipids, with more than 25,000known. Two prominent groups are the sterols, which are important membrane constituents, and the carotenoids, which collect light for photosynthesis, protect plants from excessive light, attract pollinators and fruit dispersal agents, and provide vitamin A precursors for consumers. Biotechnology has provided both a tool for understanding plant lipid metabolism, and a means of altering quality and quantity of these constituents to better serve the needs of industry and human nutrition better.

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Identification of Non-Heme Diiron Proteins That Catalyze Triple Bond and Epoxy Group Formation

Cited by 244 publications

References 13 publications

Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

The convergent evolution of defensive polyacetylenic fatty acid biosynthesis genes in soldier beetles

Lipid Metabolism

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