Petra Sperling

The evolution of desaturases

When considering the evolution of desaturases, several different aspects come into focus, the most obvious ones being phylogenetic origins and differentiation of regioselectivities of these enzymes. In this general context the term desaturase includes all enzymes able to activate oxygen and to use this reagent for a subsequent modification of C–H bonds at saturated or monounsaturated carbons in substrates as diverse as alkyl groups, acyl residues in thio-, amideor oxygen-ester linkage, carotenoids, sphingolipids, aldehydes and sterols [1,2]. The presently known oxygen-dependent modifications do not only include the formation of cisand transdouble bonds, they also result in the production of acetylenic bonds, insertion of hydroxy or epoxy groups, and even the postulated decarbonylation of aldehydes or dehydrogenation of ubiquinols [3]. This wide spectrum of reactions is catalysed by proteins which all (as extrapolated from the few examples actually studied in detail) may house a di-iron complex held in place by the side chains of suitable amino acids (histidine, aspartate, glutamate and glutamine), although some similar reactions are catalysed by the heme iron of cytochrome P450 isoforms [4]. It should also be pointed out that the mitochondrial dehydrogenation of ubiquinol by the alternative oxidase [3] does not attack a C–H–, but an O–H bond. If the activity of this enzyme does in fact rely on a di-iron centre, it seems to make use of an overpowered reagent for a reaction which normally involves the completely different di-iron–sulphur cluster of the Rieske protein.

Plant sphingolipids: structural diversity, biosynthesis, first genes and functions.

In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains. More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions. In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function. This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis. Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses.

A sphingolipid desaturase from higher plants. Identification of a new cytochrome b5 fusion protein.

A recently cloned cDNA from sunflower codes for a fusion protein composed of an N-terminal cytochromeb 5 and a domain similar to membrane-bound acyl lipid desaturases. For a functional identification, homologous cDNAs from Brassica napus and Arabidopsis thaliana were expressed in Saccharomyces cerevisiae, and sphingolipid long chain bases were analyzed. The expression of the heterologous enzyme results in significant proportions of new Δ8,9-cis/trans-phytosphingenines that accompany the residual C18-phytosphinganine predominating in wild-type yeast cells. These results represent the first identification of a gene coding for a sphingolipid desaturase and for a stereounselective desaturase showingtrans-activity from any organism. Furthermore, this fusion protein is a new member of the cytochrome b 5superfamily. The formation of the two regioisomeric phytosphingenines in the transformed yeast sheds new light on the factors controlling regioselectivity.

Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results

Abbreviations: ACP = acyl carrier protein. – ACS = acyl-CoA synthase. – ARA = arachidonic acid. – CLA = conjugated linoleic acid. – CPT = CDP-choline :1,2-diacylglycerol cholinephosphotransferase. – DAGAT = acyl-CoA :1,2-diacylglycerol acyltransferase. – DHA = docosahexaenoic acid. – EPA = eicosapentaenoic acid. – ER = endoplasmic reticulum. – FAE = fatty acid elongase. – FAR = acyl-CoA reductase. – GLA = γ-linolenic acid. – GPAT = acyl-CoA : glycerol-3-phosphate acyltransferase. – HEAR = high-erucic acid rapeseed. – KAS = β-ketoacyl-ACP synthase. – KCS = β-ketoacyl-CoA synthase. – LPAAT = acyl-CoA : lysophosphatidic acid acyltransferase. – LPCAT = acyl-CoA : lysophosphatidylcholine acyltransferase. – MCF = medium-chain fatty acids. – mRNAi = antisense messenger ribonucleic acid containing inverted-repeat sequences. – PC = phosphatidylcholine. – PDAT = phospholipid : 1,2-diacylglycerol acyltransferase. – PLA2 = phospholipase A2. – TAG = triacylglycerol. – VLCPUFA = very long-chain polyunsaturated fatty acids. – WS = wax synthase

A growing family of cytochrome b5-domain fusion proteins

The discovery of cytochrome b5-fused desaturases and hydroxylases (see Fig. 1Fig. 1) has interesting implications for both the evolution of multifunctional, multidomain enzymes and the specificity (i.e. stereochemistry, regioselectivity and substrate specificity) of desaturases. Southern blot analysis of the tobacco genome indicates that cytochrome b5 is present as a small gene family27xTobacco cytochrome b5: cDNA isolation, expression analysis, in vitro protein targeting. Smith, M.A. et al. Plant Mol. Biol. 1994; 25: 527–537Crossref | PubMed | Scopus (26)See all References27. However, plants such as B. officinalis express at least four distinct cytochrome b5-domain proteins: the ‘free’ cytochrome b5 protein, an internal domain of nitrate reductase and two different N-terminal sequences in the Δ6-acyl group- and the Δ8-sphingolipid desaturases. All of these cytochrome b5-domain sequences have significantly diverged, although without modification of the His-Pro-Gly-Gly motif involved in heme-binding9xThe cytochrome b5-fold: an adaptable molecule. Lederer, F. Biochimie. 1994; 76: 674–692Crossref | PubMed | Scopus (79)See all References, 15xA new class of cytochrome b5 fusion proteins. Napier, J.A. et al. Biochem. J. 1997; 328: 717–720PubMedSee all References. It may be that the fusion of the mobile electron carrier (cytochrome b5) to various positions on an acceptor protein provides a kinetic advantage. However, cytochrome b5 and its reductase are usually found in excess in microsomal membranes28xHigh oleic sunflower: studies on composition, desaturation of acyl groups in different lipids, organs. Sperling, P. et al. Z. Naturforsch. 1990; C45: 166–172See all References28, although this may imply that they are not particularly efficient. Another intriguing aspect, is why acyl-desaturases with Δ12- and Δ15-regioselectivities appear to lack a fused cytochrome b5 domain, even though these desaturases are much more prevalent in the plant kingdom. It may be that the presence of the b5-domain is restricted to enzymes that modify the proximal portion of lipid components facing the membrane surface (Δ2-Δ9 in acyl-CoA; O-acyl groups of glycerolipids; N-acyl groups and long-chain bases of ceramides).Fig. 1Examples of the cytochrome b5 superfamily. The position of the diagnostic cytochrome b5 heme-binding domain is indicated, although the exact positions are not to scale. The GenBank accession numbers for the sequences are: Oryza sativa cytochrome b5, X75670; Rattus norvegicus sulphite oxidase, L05084; Saccharomyces cerevisiae cytochrome b2, X03215; Lycopersicon esculentum nitrate reductase, X14060; S. cerevisiae Δ9-fatty acid desaturase (OLE1), J05676; Mortierella alpina Δ5-fatty acid desaturase, AF054824; Borago officinalis Δ6-fatty acid desaturase, U79010; Arabidopsis thaliana Δ8-sphingolipid-desaturase, AJ224161; S. cerevisiae sphingolipid-hydroxylase (FAH1), Z49260; Physcomitrella patens Δ6-fatty acid desaturase AJ222980.View Large Image | Download PowerPoint SlideThese features also call into question whether cytochrome b5 was independently fused to desaturases that had already acquired their different specificities, or whether an ancestral fusion protein for proximal lipid modification duplicated and subsequently evolved into different desaturase/hydroxylase enzymes. Phylogenetic analysis indicates that these fusion events may have happened independently at least twice, with one branch comprising the Δ5-, Δ6- and Δ8-glycerolipid/sphingolipid- desaturases (Fig. 2Fig. 2). With regard to the possible ancestral gene for this branch, it is interesting to note that Δ8-unsaturated long-chain bases are much more wide-spread in present day plants than Δ5- or Δ6-unsaturated fatty acids; this may imply that the sphingolipid-desaturase evolved first. It will be interesting to assess whether there is any change in the fitness of a plant in which the capacity to perform Δ8-sphingolipid-desaturation has been disrupted.Fig.2Phylogenetic tree analysis of cytochrome b5-fusion proteins involved in proximal fatty acid modification. Alignments were generated by the CLUSTAL-X program, and the phylogenetic tree was made with ‘TreeView’. Sequences analysed are: d9Cm=Cyanidioschyzon merolae (AB006677); d9Sc=Saccharomyces cerevisiae Δ9-fatty acid desaturase (OLE1); d5Ma=Mortierella alpina Δ5-fatty acid desaturase; d6Pp=Physcomitrella patens Δ6-fatty acid desaturase; d6Ce=Caenorhabditis elegans Δ6-fatty acid desaturase (AF031477); d5Ce=C. elegans Δ5-fatty acid desaturase (Z81122); d6Bo=Borago officinalis Δ6-fatty acid desaturase; d8Ha=Helianthus annuus Δ8-sphingolipid-desaturase (X87143); d8Bn=Brassica napus Δ8-sphingolipid-desaturase (AJ224160); d8At=Arabidopsis thaliana Δ8-sphingolipid-desaturase (AJ224161); d2Sc=S. cerevisiae sphingolipid-hydroxylase (FAH1). Accession numbers for sequences not described in Fig. 1Fig. 1 are also included.View Large Image | Download PowerPoint Slide

Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.)

In addition to the normal-type sunflower (Helianthus annuus L.) where linoleic acid is the major seed fatty acid, a dominant negative high-oleic mutant with oleic acid as the predominant fatty acid was previously obtained. We report the isolation and characterization of three different cDNA sequences, designated Ha89FAD2-1, Ha89FAD2-2, and Ha89FAD2-3, encoding sunflower microsomal oleate desaturases (FAD2), using a PCR strategy. All three deduced amino acid sequences showed significant homology to the known plant FAD2 sequences. Genomic Southern blot analysis revealed that at least one copy of each of these genes is present in the sunflower genome, except for the FAD2-1 gene from the high-oleic mutant, which might be duplicated. The FAD2-2 and FAD2-3 genes were weakly expressed in all tissues studied from both varieties. In contrast, the FAD2-1 gene was expressed strongly and exclusively in developing embryos of normal-type sunflower, whereas its expression in high-oleic developing embryos was drastically reduced. Functional expression of the corresponding cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Furthermore, the FAD2-1 gene from the high-oleic variety also expresses a fully active enzyme. These results suggest that the high-oleic mutation in sunflower interferes with the regulation of the transcription of the seed-specific FAD2 gene.

Functional Characterization of a Higher Plant Sphingolipid Δ4-Desaturase: Defining the Role of Sphingosine and Sphingosine-1-Phosphate in Arabidopsis

The role of Δ4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Δ4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Δ4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Δ4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Δ4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Δ4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Δ4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Δ4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.

A Higher Plant Δ8 Sphingolipid Desaturase with a Preference for (Z)-Isomer Formation Confers Aluminum Tolerance to Yeast and Plants

Three plant cDNA libraries were expressed in yeast (Saccharomyces cerevisiae) and screened on agar plates containing toxic concentrations of aluminum. Nine cDNAs were isolated that enhanced the aluminum tolerance of yeast. These cDNAs were constitutively expressed in Arabidopsis (Arabidopsis thaliana) and one cDNA from the roots of Stylosanthes hamata, designated S851, conferred greater aluminum tolerance to the transgenic seedlings. The protein predicted to be encoded by S851 showed an equally high similarity to Δ6 fatty acyl lipid desaturases and Δ8 sphingolipid desaturases. We expressed other known Δ6 desaturase and Δ8 desaturase genes in yeast and showed that a Δ6 fatty acyl desaturase from Echium plantagineum did not confer aluminum tolerance, whereas a Δ8 sphingobase desaturase from Arabidopsis did confer aluminum tolerance. Analysis of the fatty acids and sphingobases of the transgenic yeast and plant cells demonstrated that S851 encodes a Δ8 sphingobase desaturase, which leads to the accumulation of 8(Z/E)-C18-phytosphingenine and 8(Z/E)-C20-phytopshingenine in yeast and to the accumulation of 8(Z/E)-C18-phytosphingenine in the leaves and roots of Arabidopsis plants. The newly formed 8(Z/E)-C18-phytosphingenine in transgenic yeast accounted for 3 mol% of the total sphingobases with a 8(Z):8(E)-isomer ratio of approximately 4:1. The accumulation of 8(Z)-C18-phytosphingenine in transgenic Arabidopsis shifted the ratio of the 8(Z):8(E) isomers from 1:4 in wild-type plants to 1:1 in transgenic plants. These results indicate that S851 encodes the first Δ8 sphingolipid desaturase to be identified in higher plants with a preference for the 8(Z)-isomer. They further demonstrate that changes in the sphingolipid composition of cell membranes can protect plants from aluminum stress.

Desaturases fused to their electron donor

Fatty acid desaturations in the carboxy-terminal segment from C1-C10 are catalyzed in many, but not in all cases, by desaturase enzymes which are fused to their electron donor cytochrome b 5 . Several of these enzymes (front-end desaturases) from a wide variety of organisms have been cloned and functionally expressed for proof of regio-, stereo- and chain length-selectivity. In most cases the actual status of the substrate chain, whether coenzyme A thioester or component of a membrane lipid, is not known. The cytochrome b 5 domain is located N-terminally, internally or C-terminally. Compared to the free cytochrome b 5 , the fused domains show a significant reduction of acidic amino acid residues on the surface of the four helices enclosing the heme group. It is discussed how this may contribute to hydrophobic domain pairing required for interdomain electron transport. This is in contrast to the mode of interaction of free cytochrome b 5 with its partners, which is governed by electrostatic charge pairing. A look at crystallized or computer-simulated models involving fused or free cytochrome b 5 helps to outline the problems encountered by optimizing the docking of partners and the exchange of electrons between domains of different degrees of mobility.

Identification of Fungal Sphingolipid C9-methyltransferases by Phylogenetic Profiling

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24–71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Δ4,8-desaturated ceramide as substrates.