Research paperDelta or Omega? Δ12 (ω6) fatty acid desaturases count 3C after the pre-existing double bond
Introduction
Polyunsaturated ω3 and ω6 FAs belong to the “essential fats” consumed by animals with a diet. All living organisms synthesize monounsaturated palmitoleic (16:1Δ9) or oleic (18:1Δ9) acids from fully saturated precursors, palmitic (16:0) and stearic (18:0) acids, respectively, employing the oxygen-dependent dehydrogenation by soluble or integral membrane Δ9-FADs. Dienoic (diunsaturated) linoleic (18:2Δ9,12) acid is the product of desaturation of oleic acid by the acyl-lipid Δ12-FADs (sometimes referred to as ω6-FADs), which are conservative in structure from bacteria to plants and lower animals [[1], [2], [3], [4]]. Further desaturation of linoleic acid by a Δ15-FAD in algae, fungi, and plants results in the formation of trienoic α-linolenic (18:3Δ9,12,15) acid – the first member of ω3 essential FAs, which may be further elongated and desaturated (mainly, in algae, fungi and some Protista) into important precursors of the icosanoids − C20-22 FAs, including leukotrienes, prostaglandins, thromboxanes, etc.
FADs are highly specific towards the length of their acyl substrates, as well as to the position and geometric configuration of the newly introduced cis double bonds [4]. The counting mode of FADs (from a carboxyl (Δ) or a methyl (ω) terminus) is a long-standing question with yet no clear answer [5]: in monounsaturated oleic acid a single double bond may be equally assigned to Δ9 or ω9 positions. The counting mode of plant-type soluble Acyl-Carrier-Protein (ACP) desaturases was experimentally determined relative to the carboxyl end of the FA [6,7] (Δ positioning). Instead, integral acyl-lipid FADs developed, at least, three distinct methods of double bonds positioning. (1) A double bond is introduced between specific carbon atoms counted from the carboxyl terminus (Δ) or (2) methyl terminus (ω) of the FA chain, and (3) a subsequent double bond is introduced 3 carbons (3C) from a pre-existing double bond (x + 3) [4]. It is considered that these FAD regioselectivities are not mutually exclusive and may be assigned as primary and secondary modes of FADs activities [8].
Unlike eukaryotic plants that carry soluble acyl-ACP FADs (mostly Δ9) and other integral membrane acyl-lipid FADs (including Δ12-and Δ15-FADs of different subcellular localization), prokaryotic cyanobacteria (one of the most ancient group of organisms on Earth) possess only acyl-lipid FADs that introduce all double bonds into acyl chains bound to membrane lipids [9]. In vivo studies of FA desaturation and elongation in the cyanobacterium Synechocystis sp. strain PCC 6803 supplied with heptanoic acid (C7) suggested that the primary Δ9-FAD (that converts saturated palmitic or stearic acids to monounsaturated palmitoleic or oleic acids) counts from the carboxyl terminus of a FA, while the terminal Δ15-FAD counts from the methyl terminus, representing, in fact, ω3-FAD [10]. Although it was suggested that Δ12-FAD counts from a C-terminus, the counting mechanisms of this enzyme was not fully understood. In higher plants, plastid and microsomal Δ12-FADs, as well as fungal microsomal Δ12-FADs, are also represented by acyl-lipid desaturases with proposed similar mechanism of action. Here we present the evidence that prokaryotic cyanobacterial acyl-lipid Δ12-FADs desaturate FAs counting 3C toward the methyl end from an existing double bond in the monoene, thus representing the (ν + 3)-type FADs.
Section snippets
Cyanobacterial strains and growth conditions
The model cyanobacteria strains, Synechocystis sp. PCC 6803 (sub-strain GT) and Synechococcus elongatus PCC 7942, as well as the unusual cyanobacterial strain, Gloeobacter violaceus PCC 7421, which lacks the thylakoid membranes, have been used in the experiments. Cell cultures were maintained on solid BG-11 medium [11] supplemented with 1.2% agar. For the experiments, cyanobacterial culture was aseptically transferred into the liquid BG-11 medium buffered with 20 mM HEPES-NaOH pH 7.5 and grown
Expression of Δ12-FAD from Gloeobacter in Synechococcus
The desA genes that code for Δ12-FADs have been cloned from the unusual cyanobacterium Gloeobacter violaceus PCC 7421 (glr2623) [21], which does not have thylakoid membranes, and from the model Synechocystis strain sp. PCC 6803 (slr1350) [22]. It should be noted that, unlike Synechocystis that has only one desA gene, the genome of G. violaceus carries two genes homologous to desA, glr2623 and gll3735 (Supplementary Fig. S1). Our attempts to express the gll3735 gene in E. coli or in Synechococcus
Discussion
Previously, the plant fad-2 gene (Δ12-FAD of the endoplasmic reticulum) of peanut (Arachis hypogaea L.) was expressed in the unicellular eukaryotic yeast, Saccharomyces cerevisae [27] that, similarly to prokaryotic Synechococcus elongatus, carries only one Δ9-FAD, (not acyl-lipid, but acyl-CoA FAD) and synthesizes only monounsaturated FAs [28]. Transformed yeast synthesized 18:2Δ9,12 from 18:1Δ9, and 16:2Δ9,12 from 16:1Δ9 in a standard growth medium. Supplementation of a growth medium with cis
Author contribution
AYS performed plasmid construction and gene expression; RAS analyzed lipids, fatty acids, and prepared figures; KSM participated in gene cloning and analyzed fatty acids; SVG performed ESI MS and analyzed the data; DAL supervised the research and wrote the article.
Funding information
This work was supported, in part, by The Ministry of Science and Higher Education of the Russian Federation (Project no. 0106-2019-0010) and Russian Science Foundation (grants no. 14-24-00020 to D.A.L and 19-74-10100 to K·S.M.). Low-energy electrospray ionization mass spectrometry studies were supported by the RUDN University Program 5–100.
Declaration of competing interest
Authors declare no conflict of interests.
Acknowledgements
In this work the large-scale research facilities of the Collection of microalgae and cyanobacteria IPPAS (K.A. Timiryazev Institute of Plant Physiology RAS, Moscow, Russia) were used.
References (34)
- et al.
Classification and substrate head-group specificity of membrane fatty acid desaturases
Comput. Struct. Biotechnol. J.
(2016) Palmitoleate formation by soybean stearoyl-acyl carrier protein desaturase
Biochim. Biophys. Acta
(1993)- et al.
Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea
J. Biol. Chem.
(2007) - et al.
Structure and expression of fatty acid desaturases
Biochim. Biophys. Acta
(1998) Isolation and purification of cyanobacteria
Methods Enzymol.
(1988)Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803
Methods Enzymol.
(1988)- et al.
Application of bioluminescence to the study of circadian rhythms in cyanobacteria
Methods Enzymol.
(2000) - et al.
Light-dependent cold-induced fatty acid unsaturation, changes in membrane fluidity, and alterations in gene expression in Synechocystis
Biochim. Biophys. Acta
(2012) - et al.
Beta-ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis
J. Biol. Chem.
(1980) - et al.
Endoplasmic oleoyl-PC desaturase references the second double bond
Phytochemistry
(2001)
Structural determinant of functionality in acyl lipid desaturases
J. Lipid Res.
Regulatory role of membrane fluidity in gene expression and physiological functions
Photosynth. Res.
Control of fatty acid desaturation: a mechanism conserved from bacteria to humans
Mol. Microbiol.
Cyanophage-encoded lipid desaturases: oceanic distribution, diversity and function
ISME J.
Desaturation and related modifications of fatty acids
Annu. Rev. Plant Physiol. Plant Mol. Biol.
Metabolic evidence for the involvement of a Δ4-palmitoyl-acyl carrier protein desaturase in the synthesis of petroselinic acid in coriander endosperm and transgenic tobacco cells
Plant Physiol.
An in vivo study of substrate specificities of acyl-lipid desaturases and acyltransferases in lipid synthesis in Synechocystis PCC6803
Plant Physiol.
Cited by (7)
Effect of Desaturase Gene Overexpression on Fatty Acid Synthesis in Escherichia coli
2024, Shipin Kexue/Food ScienceThe Specificities of Lysophosphatidic Acid Acyltransferase and Fatty Acid Desaturase Determine the High Content of Myristic and Myristoleic Acids in Cyanobacterium sp. IPPAS B-1200
2024, International Journal of Molecular SciencesEfficacy and Biomedical Roles of Unsaturated Fatty Acids as Bioactive Food Components
2023, Current Chemical BiologyCatalytic mechanisms underlying fungal fatty acid desaturases activities
2023, Critical Reviews in Biotechnology