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Classification and substrate head-group specificity of membrane fatty acid desaturases.

ABSTRACT: Membrane fatty acid desaturases are a diverse superfamily of enzymes that catalyze the introduction of double bonds into fatty acids. They are essential in a range of metabolic processes, such as the production of omega-3 fatty acids. However, our structure-function understanding of this superfamily is still developing and their range of activities and substrate specificities are broad, and often overlapping, which has made their systematic characterization challenging. A central issue with characterizing these proteins has been the lack of a structural model, which has been overcome with the recent publication of the crystal structures of two mammalian fatty acid desaturases. In this work, we have used sequence similarity networks to investigate the similarity among over 5000 related membrane fatty acid desaturase sequences, leading to a detailed classification of the superfamily, families and subfamilies with regard to their function and substrate head-group specificity. This work will facilitate rapid prediction of the function and specificity of new and existing sequences, as well as forming a basis for future efforts to manipulate the substrate specificity of these proteins for biotechnology applications.

SUBMITTER: Li D

PROVIDER: S-EPMC5037126 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

Project description:Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat ?6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of ?5 desaturase (D5d), which converts dihomo-?-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-?-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish ?5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (?9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids.

| S-EPMC4689341 | biostudies-literature

Expression and purification of integral membrane fatty acid desaturases.

Project description:Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.

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Comparison of the Substrate Preferences of ?3 Fatty Acid Desaturases for Long Chain Polyunsaturated Fatty Acids.

Project description:Omega-3 long chain polyunsaturated fatty acids (?3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5?3) and docosahexaenoic acid (DHA; 22:6?3) are important fatty acids for human health. These ?3 LC-PUFAs are produced from their ?3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from ?6 LC-PUFA by omega-3 desaturases (?3Ds). Here, we have investigated eight ?3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 ?6 PUFA substrates by transiently expressing them in Nicotiana benthamiana leaves. Our results showed hitherto unreported activity of many of the ?3Ds on ?6 LC-PUFA substrates leading to their conversion to ?3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.

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Substrate specificity and membrane topologies of the iron-containing ?3 and ?6 desaturases from Mortierella alpina.

Project description:Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ?6 and ?3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl-coenzyme A desaturase-1 (SCD1) and the related sphingolipid ?-hydroxylase (Scs7p) that act upon different substrates.

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Acinetobacter baumannii Fatty Acid Desaturases Facilitate Survival in Distinct Environments

Project description:RNA sequencing transcriptomics was performed on a highly multidrug resistant A. baumannii strain belonging to international clone I, AB5075_UW and a transposon insertion inactivated mutant of ABUW_1103 (DesT1), which encodes for a TetR family regulator. Transcriptomics revealed that DesT1 might function as a global regulator controlling expression of various genes involved in fatty acid metabolism and transport and a range of outer membrane proteins. Genes encoding acyl-CoA desaturase and ferredoxin reductase were found to be significantly upregulated when DesT1 regulator was inactivated. Aerobic desaturases are known to play an important role in maintaining membrane homeostasis.

2021-06-15 | GSE163107 | GEO

Comparative analysis of fatty acid desaturases in cyanobacterial genomes.

Project description:Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous or N(2)-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteria Synechococcus and Prochlorococcus differs from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. Strain Gloeobacter violaceus PCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, lack highly unsaturated fatty acids in lipids and contain only one Delta9 desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution.

| S-EPMC2593844 | biostudies-literature

Identification and characterization of new ?-17 fatty acid desaturases.

Project description:?-3 fatty acid desaturase is a key enzyme for the biosynthesis of ?-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ?-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ?-3 desaturases share 55 % identity at the amino acid level with the known ?-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional ?-12/?-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ?-6 arachidonic acid to the ?-3 eicosapentanoic acid, with a substrate conversion efficiency of 54-65 %. These enzymes have a broad ?-6 fatty acid substrate spectrum, including both C18 and C20 ?-6 fatty acids although they prefer the C20 substrates, and have strong ?-17 desaturase activity but weaker ?-15 desaturase activity. Thus, they belong to the ?-17 desaturase class. Unlike the previously identified bifunctional ?-12/?-15 desaturase from F. monoliforme, they lack ?-12 desaturase activity. The newly identified ?-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these ?-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ?-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

| S-EPMC3570762 | biostudies-literature

Biological Role of Unsaturated Fatty Acid Desaturases in Health and Disease.

Project description:Polyunsaturated fatty acids (PUFAs) are considered one of the most important components of cells that influence normal development and function of many organisms, both eukaryotes and prokaryotes. Unsaturated fatty acid desaturases play a crucial role in the synthesis of PUFAs, inserting additional unsaturated bonds into the acyl chain. The level of expression and activity of different types of desaturases determines profiles of PUFAs. It is well recognized that qualitative and quantitative changes in the PUFA profile, resulting from alterations in the expression and activity of fatty acid desaturases, are associated with many pathological conditions. Understanding of underlying mechanisms of fatty acid desaturase activity and their functional modification will facilitate the development of novel therapeutic strategies in diseases associated with qualitative and quantitative disorders of PUFA.

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Selection in Europeans on Fatty Acid Desaturases Associated with Dietary Changes.

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| S-EPMC5435082 | biostudies-literature

Inventory of fatty acid desaturases in the pennate diatom Phaeodactylum tricornutum.

Project description:The diatom Phaeodactylum is rich in very long chain polyunsaturated fatty acids (PUFAs). Fatty acid (FA) synthesis, elongation, and desaturation have been studied in depth in plants including Arabidopsis, but for secondary endosymbionts the full picture remains unclear. FAs are synthesized up to a chain length of 18 carbons inside chloroplasts, where they can be incorporated into glycerolipids. They are also exported to the ER for phospho- and betaine lipid syntheses. Elongation of FAs up to 22 carbons occurs in the ER. PUFAs can be reimported into plastids to serve as precursors for glycerolipids. In both organelles, FA desaturases are present, introducing double bonds between carbon atoms and giving rise to a variety of molecular species. In addition to the four desaturases characterized in Phaeodactylum (FAD2, FAD6, PtD5, PtD6), we identified eight putative desaturase genes. Combining subcellular localization predictions and comparisons with desaturases from other organisms like Arabidopsis, we propose a scheme at the whole cell level, including features that are likely specific to secondary endosymbionts.

| S-EPMC4377986 | biostudies-literature

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